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Vascular-cognitive impairment after chronic high-thoracic spinal cord injury Jia, Mengyao 2020

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  Vascular-cognitive Impairment after Chronic High-thoracic Spinal Cord Injury  by  Mengyao Jia  B.Sc., University of British Columbia, 2015    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE  in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)          THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2020   © Mengyao Jia, 2020  ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled:  Vascular-cognitive Impairment after Chronic High-thoracic Spinal Cord Injury  submitted by Mengyao Jia  in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine   Examining Committee: Dr. Andrei V. Krassioukov, Professor, Department of Medicine, UBC Supervisor  Dr. Ismail Laher, Professor, Department of Anesthesiology, Pharmacology & Therapeutics, UBC Supervisory Committee Member  Dr. Aaron A. Phillips, Assistant Professor, Department of Physiology & Pharmacology, University of Calgary Supervisory Committee Member Dr. Fan Fan, Assistant Professor, Department of Pharmacology and Toxicology, University of Mississippi Additional Examiner   iii  Abstract Individuals living with chronic spinal cord injury (SCI) often exhibit impairments in cognitive function, which impedes their rehabilitation and possible transition into community. While a number of clinical studies have demonstrated the impact of impaired cardiovascular control on cognitive impairment, the mechanistic understanding of this deleterious relationship is still lacking. The present study investigates whether chronic disruption of cardiovascular control following experimental SCI results in cerebrovascular decline and vascular cognitive impairment. Fourteen weeks following a high thoracic SCI (at 3rd thoracic segment), rats were subjected to a battery of in vivo and in vitro physiological assessments, cognitive-behavioral tests, and immunohistochemical approaches to investigate changes in cerebrovascular structure and function in middle cerebral artery (MCA). We show that in MCA of rats with SCI, there is 55% reduction in the maximal vasodilator response to carbachol, which is associated with reduced expression of endothelial marker cluster of differentiation 31 (CD31) and transient receptor potential cation channel 4 (TRPV4) channels. Compared to controls, MCAs in rats with SCI were found to have 50% more collagen I in the media of vascular wall and 37% less distensibility at physiological intralumenal pressure. Furthermore in SCI group, the cerebral blood flow (CBF) in the hippocampus was reduced by 32% in association with impairment in short-term memory based on a novel object recognition test. There were no changes in the sympathetic innervation of the vasculature and passive structure in the SCI group. Chronic experimental SCI is associated with structural alteration and endothelial dysfunction in cerebral arteries that likely contributes to significantly reduced CBF and vascular cognitive impairment.   iv  Lay Summary This thesis project is designed to investigate the cognitive function of individuals living with chronic spinal cord injury (SCI). Previous clinical studies have shown that cognitive impairment is common seen among SCI patients and are associated with various factors including co-occurring concussion, vascular dysfunction, pain, lack of activity, sleep apnea etc. We created an animal model to specifically look at the effect of uncontrolled blood pressure to the brain vasculature and whether it leads to memory loss. The results demonstrated that 14 weeks after the injury, animals demonstrated impaired short-term memory, together with dropped brain blood flow and damaged brain vascular function. We conclude that the maintaining the steady control of blood pressure is crucial to prevent the cognitive impairment found after spinal cord injury.             v  Preface All of the work presented henceforth was conducted in the Autonomic Dysfunction Laboratory at International Collaboration On Rehabilitation Discoveries (ICORD). All experiments were approved by the University of British Columbia’s Animal Care Committee under the certificate A14-0152. Chapter I has been published as Phillips, A. A., N. Matin, B. Frias, M. M. Z. Zheng, M. Jia, C. West, A. M. Dorrance, I. Laher, and A. V. Krassioukov. “Rigid and Remodelled: Cerebrovascular Structure and Function After Experimental high‐thoracic Spinal Cord Transection.” The Journal of Physiology 594, no. 6 (2016): 1677-1688.  Dr. Krassioukov was the principle investigator and Dr. Phillips led the project. Dr. Matin and Dr. Dorrance were the collaborators on the pressure myography experiments. Dr. Frias was responsible of the surgeries and provided guidance of the immunohistochemitry experiments. Ms. Zheng was involved in the animal care. All authors were involved in manuscript editing. I performed the immunohistochemistry and provided edits to the manuscript. Chapter II has been published as Aaron A. Phillips, Nusrat Matin, Mengyao Jia, Jordan W. Squair, Aaron Monga, Mei Mu Zi Zheng, Rahul Sachdeva, Andrew Yung, Shea Hocaloski, Stacy Elliott, Piotr Kozlowski, Anne M. Dorrance, Ismail Laher, Philip N. Ainslie, and Andrei V. Krassioukov. “Transient Hypertension after Spinal Cord Injury Leads to Cerebrovascular Endothelial Dysfunction and Fibrosis.” Journal of Neurotrauma 35:573–58. Dr. Krassioukov was the principle investigator and Dr. Phillips led the project. Dr. Matin, Dr. Laher and Dr. Dorrance were the collaborators on the pressure myography experiments. Mr. Yung and Dr. Kozlowski were the collaborators on the MRI studies. Ms. Hocaloski, Dr. Elliott, and Dr. Ainslie were the collaborators on the human studies. Mr. Squair was responsible of the ultrasound experiments.  Dr. vi  Sachdeva was responsible of the surgeries and provided guidance of the immunohistochemitry experiments. Ms. Zheng and Mr. Monga were involved in the animal care. I was involved in animal care and experimental treatments, also performed the immunohistochemistry and manuscript editing.  Chapter III has been accepted to Journal of Neurotrauma as “Vascular-Cognitive Impairment Following High-Thoracic Spinal Cord Injury is Associated with Structural and Functional Maladaptations in Cerebrovasculature” (in press). Authors of the manuscripts are: Mengyao Jia*, Rahul Sachdeva*, Aaron A. Phillips, Shaoxun Wang, Andrew Yung, Mei Mu Zi Zheng, Sarah Leong, Piotr Kozlowski, Richard J. Roman, Andrei V. Krassioukov. *co-first author. I was the lead investigator for chapter III study, responsible for all major areas of concept formation, surgery, data collection and analysis, as well as manuscript composition. Ms. Mei Mu Zi Zheng was involved in animal care and experimental treatments. Novel object recognition tests were conducted with the help from Ms. Amanda Lee.  In vitro pressure myography was performed by me and Mr. Shaoxun Wang together under guidance from Dr. Roman. Arterial spin labelling MRI scanning was performed by Mr. Andrew Yung at UBC MRI center under guidance from Dr. Kozlowski. Tissue harvesting and processing for downstream applications and immunohistochemistry was carried out by myself. Data extraction and analyses was conducted by me with help from Ms. Sarah Leong and Karina Chornanka. Dr. Rahul Sachdeva and I drafted this manuscript, with edits by Dr. Andrei Krassioukov and Dr. Aaron Phillips.          vii  Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface ............................................................................................................................................ v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1. Cerebrovascular structure and function after experimental high‐thoracic spinal cord transection ............................................................................................................................. 1 1.1 Introduction ........................................................................................................................... 1 1.2 Methods................................................................................................................................. 3 1.2.1 Surgery and animal care ................................................................................................. 3 1.2.2 Pressure myography studies ........................................................................................... 4 Reactivity of the MCA ............................................................................................................ 5 1.2.3 Immunohistochemical analyses ..................................................................................... 5 1.2.4 Immunohistochemical morphometric assessment of the MCA ..................................... 7 viii  1.2.5 TH analysis of MCA whole mounts .............................................................................. 7 1.2.6 Statistical analyses ......................................................................................................... 8 1.3 Results ................................................................................................................................... 9 1.3.1 Physiological parameters ............................................................................................... 9 1.3.2 SCI detrimentally altered structure of the MCA ............................................................ 9 1.4 Discussion ........................................................................................................................... 19 1.5 Conclusions ......................................................................................................................... 24 Chapter 2. Transient cerebral hyper-perfusion due to autonomic dysreflexia impairs cerebrovascular function and structure .................................................................................... 25 2.1 Introduction ......................................................................................................................... 25 2.2 Methods............................................................................................................................... 27 2.2.1 Experimental protocol. ................................................................................................. 27 2.2.2 Urodynamic assessments. ............................................................................................ 28 2.2.3 Penile vibrostimulation for sperm retrieval. ................................................................ 28 2.2.4 Clinical cardiovascular and cerebrovascular assessments. .......................................... 28 2.2.5 Experimental protocol. ................................................................................................. 29 2.2.6 Pre-clinical spinal cord transection. ............................................................................. 30 2.2.7 Ultrasound evaluation of middle cerebral artery blood flow with concurrent blood pressure during acute AD/transient hypertension induced by colorectal distension. ........... 31 2.2.8 Repetitive colorectal distension to induce AD. ............................................................ 32 2.2.9 Reactivity of the MCA. ................................................................................................ 33 2.2.10 Immunohistochemical analyses. ................................................................................ 33 ix  2.2.11 Tyrosine hydroxylase (TH) analysis of MCAs whole mounts. ................................. 34 2.2.12 Morphological assessment of MCA. .......................................................................... 34 2.2.13 Molecular biology assessment of the MCA. .............................................................. 34 2.2.14 CBF ............................................................................................................................ 35 2.2.15 Statistical analyses. .................................................................................................... 36 2.3 Results ................................................................................................................................. 36 2.3.1 Clinical evaluation during transient hypertension. ...................................................... 36 2.3.2 Pre-clinical model of transient hypertension ............................................................... 36 2.2.4 Cerebrovascular consequences of chronic transient hypertension ............................... 37 2.4 Discussion ........................................................................................................................... 38 2.5 Clinical perspectives ........................................................................................................... 41 Chapter 3. Vascular-cognitive impairment following high-thoracic spinal cord injury is associated with structural and functional maladaptation in cerebrovasculature ................. 50 3.1 Introduction ......................................................................................................................... 50 3.2 Methods............................................................................................................................... 51 3.2.1 Experimental design ..................................................................................................... 51 3.2.3 T3 Transection surgery and animal care ...................................................................... 52 3.2.4 NORT ........................................................................................................................... 53 3.2.5 CBF .............................................................................................................................. 54 3.2.6 Pressure myography ..................................................................................................... 54 3.2.7 Perfusion and tissue collection ..................................................................................... 55   x  3.2.8 Immunohistochemistry ................................................................................................ 56 Quantification ....................................................................................................................... 56 3.2.9 Morphometric assessment of the MCA ....................................................................... 57 3.2.10 Tyrosine hydroxylase (TH) staining of the MCA whole mounts .............................. 57 3.2.11 Statistical analyses ..................................................................................................... 57 3.3 Results ................................................................................................................................. 58 3.3.1 Altered endothelial function and mechanical properties of the MCA after chronic SCI............................................................................................................................................... 58 3.3.2 Morphological and structural changes in the MCA after SCI ..................................... 58 3.3.3 Chronic SCI is associated with a reduction in CBF ..................................................... 58 3.3.4 SCI disrupts short-term memory .................................................................................. 59 3.4 Discussion ........................................................................................................................... 59 3.5 Future perspectives ............................................................................................................. 61 References .................................................................................................................................... 69  xi   List of Tables Table 1-1. Ex vivo pressure myography data showing remodelling of the MCA after T3 spinal cord injury. ................................................................................................................................ 13 Table 2-1. List of primary antibodies used for morphological assessment of middle cerebral arteries. ...................................................................................................................................... 43 xii  List of Figures Figure 1-1. Complete T3‐SCI led to inward remodelling of the MCA .................................... 10 Figure 1-2. T3‐SCI results in hypertrophic inward remodelling of the MCA .......................... 11 Figure 1-3. Mechanical properties of the MCA after T3‐SCI .................................................. 12 Figure 1-4. Endothelial function of the MCA after SCI ........................................................... 15 Figure 1-5. Profibrotic middle cerebral artery after SCI ........................................................... 16 Figure 1-6. TH+ axonal density and fluorescence intensity after T3‐SCI ................................ 18 Figure 2-1. Developing a clinically-relevant preclinical model of transient cerebral hyper-perfusion secondary to autonomic dysreflexia. ........................................................................ 44 Figure 2-2. Chronic exposure to transient cerebral hyper-perfusion impaired middle cerebral artery endothelial function, exacerbated stiffening, and led to hyper-sensitivity to constrictor stimuli after SCI. ....................................................................................................................... 46 Figure 2-3. Endothelial mechanoreceptor overexpression, profibrosis, and loss of sympathetic perivascular innervation density in response to chronic exposure to repetitive transient hypertension. ............................................................................................................................. 48 Figure 3-1. Experimental timeline. ........................................................................................... 65 Figure 3-2. Carbachol-induced relaxation is diminished in MCA after chronic SCI, which is closely associated with the loss of TRPV 4 and CD31 staining in the endothelium. ............... 64 Figure 3-3. Chronic SCI leads to increased collagen deposition in the arterial wall. ............... 66 xiii  Figure 3-4. Reduced baseline CBF and impaired spatial short-term memory are observed after prolonged high-thoracic SCI. .................................................................................................... 67 xiii  Acknowledgements I offer my enduring gratitude to the faculty, staff and my fellow students at the UBC, who have inspired me to continue my work in this field. I owe particular thanks to Dr. Andrei Krassioukov, whose knowledge and expertise in the area of spinal cord injury have been such a tremendous benefit for me. I am so grateful for his encouragement and understanding during the toughest of times. He is and always will be a role model for me in my future endeavours.  I thank Dr. Aaron Phillips for giving me the opportunity to explore the research field. He has truly seen me grown in the past five years. I may not express this often enough, but I truly appreciate his guidance throughout my academic career. I would also like to thank my committee members Dr. Ismail Laher for his guidance and support. His dedication to research and passion for teaching has inspired me to pursue research.    Special thanks to Dr. Rahul Sachdeva for supporting me through the hardest time of my life. My project wouldn’t complete without his help and guidance.  Also thank Dr. Piotr Kozlowski, Andrew Yung, Dr. Roman, Dr. Fan Fan and Shaoxun Wang for their amazing collaboration on this project. A heartfelt thank you goes out to Annie Zheng, Amanda Lee, Sarah Leong, Aaron Monga for their help throughout the project. Thank you to those who encouraged me through some very busy months. It would not have been possible without you.  Special thanks are owed to my parents, whose have supported me throughout my years of education, both morally and financially. xiv  Dedication To my lovely Waffle and Ronnie for keeping me company   1 Chapter 1. Cerebrovascular structure and function after experimental high‐thoracic spinal cord transection   1.1 Introduction Over the past two decades, we have come to appreciate that cardiovascular disease is a primary cause of morbidity and mortality after spinal cord injury (SCI) (DeVivo et al. 2002; Garshick et al. 2005). Although a great deal of insight has been provided over that time illustrating cardiac as well as systemic vascular decline in the SCI population, there is scant literature regarding cerebrovascular function, and the associated consequences, in this population. Understanding cerebrovascular health after SCI is critical. Those with high‐thoracic or cervical SCI are 300–400% more likely to suffer stroke as compared to uninjured counterparts, even after controlling for major confounding risk factors (Wu et al. 2012; Cragg et al. 2013). In fact, those with SCI have a similarly elevated risk of stroke as able‐bodied smokers do of suffering a myocardial infarction (Yusuf et al. 2004). Also, cognitive dysfunction is widespread after SCI, which is probably mediated in part by cerebrovascular impairments (Davidoff et al. 1990; Jegede et al. 2010; Wecht & Bauman, 2013; Phillips et al. 2014 b). We have recently shown in two human clinical trials that SCI results in cerebrovascular dysfunction, which was only partially restored when increasing blood pressure to able‐bodied normal levels, indicating other important factors are playing a significant role (Phillips et al. 2014 a,b). A more direct analysis of potential additional factors contributing to impaired cerebrovascular function (e.g. remodelling, stiffness, endothelial function, vasoconstrictive reactivity, profibrosis)  2 after SCI is required to clearly elucidate the pathophysiology underlying cerebrovascular events in this population. For example, arterial remodelling, such as increased collagen and decreased elastin in arterial walls, occurs in response to profibrotic signalling [i.e. transforming growth factor β (TGF‐β)] in models of volume unloading, physical inactivity and increased engagement of the renin–angiotensin system (RAS) (Zhang et al. 2001; Satoh et al. 2001; Tuday et al. 2009; Alessandri et al. 2010; Sofronova et al. 2015). These models of dysfunction also lead to increased stiffness and alterations in arterial responsiveness to vasoactive signals (Geary et al. 1998; Tuday et al. 2009; Sofronova et al. 2015). After SCI, there is a combination of volume unloading, physical inactivity and increased reliance of the RAS system, which therefore may predispose to diverse cerebrovascular dysfunctions (Wecht et al. 2005; Handrakis et al. 2009; Groothuis & Thijssen, 2010). A fastidious understanding of cerebrovascular health after SCI will help guide interventions focused on mitigating the significant stroke burden, cognitive deficits and dysfunctional cerebral blood flow regulation that are commonly identified in those living with SCI. To date, no direct experimental examinations of cerebrovascular structure and function after SCI have been made. Herein, we have performed the first direct assessment of cerebrovascular function and structure in SCI rodents including: ex vivo pressure myography for passive structure, vasoconstrictor sensitivity and endothelial dilatation, as well as histological techniques to provide insight into the mechanisms underlying declining vascular health (i.e. collagen/elastin/TGF‐β, morphometrics). We hypothesized that high‐thoracic SCI would lead to impaired cerebrovascular function and structure, which would be associated with a profibrotic environment in the middle cerebral artery (MCA).  3 1.2 Methods Experiments were initially conducted on 25 male Wistar rats (age 9 weeks, mass 250–350 g; Harlan Laboratories, Indianapolis, IN, USA). Animals were assigned to either: sham injured control (Sham; n = 10) or T3 complete spinal cord transection groups (T3‐SCI; n = 15). Seven weeks after T3‐SCI, MCA passive structure, endothelial function and vasoconstrictive responsiveness were assessed using pressure myography, as this is of analogous duration to that which is considered ‘chronic’ in the clinical SCI population (Krassioukov & Claydon, 2006). 1.2.1 Surgery and animal care Surgery and animal care were conducted according to standard procedures in our laboratory (Alan et al. 2010; Ramsey et al. 2010). Animals received prophylactic enrofloxacin [Baytril; 10 mg kg−1, s.c., Associated Veterinary Purchasing (AVP), Langley, Canada] for 3 days prior to SCI surgery. Moreover, for 5 days prior to surgery T3‐SCI animals were provided enhanced caloric provision (i.e. Ensure, extra fruit, cereal) to ensure survival during weight loss, secondary to recovery. As such, body weight was elevated in the T3‐SCI group prior to surgery (272.6 ± 5.6 vs. 323.3 ± 3.9 g; P < 0.05). On the day of surgery, enrofloxacin (10 mg kg−1, s.c.), buprenorphine (Temgesic; 0.02 mg kg−1, s.c., McGill University) and ketoprofen (Anafen, 5 mg kg−1, s.c., AVP) were administered pre‐operatively. Rats were anaesthetized with ketamine hydrochloride (70 mg kg−1, i.p.; Vetalar; AVP) and dexmedetomidine (0.25 mg kg−1, i.p.; Domitor; AVP). A dorsal midline incision was made in the superficial muscle overlying the C7–T3 vertebrae. The dura was opened at the T2–T3 intervertebral gap and the spinal cord was completely transected at the caudal portion of the intervertebral gap using microscissors. Complete transection was confirmed by two surgeons via visual separation of the rostral and caudal spinal cord stumps, and Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, USA) was placed between the stumps to achieve haemostasis. The muscle and skin were closed with 4–0 Vicryl and  4 4–0 Prolene sutures, respectively. Animals received warmed lactated Ringer's solution (5 ml, s.c.) and recovered in a temperature‐controlled environment (Animal Intensive Care Unit, Los Angeles, CA, USA), after being administered atipamezole (1 mg kg−1, s.c., AVP) for reversal of dexmedetomidine. Enrofloxacin (10 mg kg−1, s.c.), buprenorphine (0.02 mg kg−1, s.c.) and ketoprofen (5 mg kg−1, s.c.) were administered once a day for 3 days post‐operatively. The bladder was manually expressed three to four times daily until spontaneous voiding returned (about 10 days after injury). All assessments and analyses were completed blinded of each condition/group. 1.2.2 Pressure myography studies  Seven weeks after injury, Sham and T3‐SCI rats were anaesthetized with 3% isoflurane and sacrificed by decapitation after exsanguination for ex vivo pressure myography studies. MCA function and structure were assessed by pressure myography (Living Systems, St. Albans, VT, USA) as described previously (Pires et al. 2011). A branch‐free segment of the MCA was isolated and mounted between two glass cannulas in a pressure myograph chamber, which was then placed in an inverted microscope coupled to a camera (Olympus SZ30, Centre Valley, PA, USA) and autodetection unit (Living Systems, VDA‐10) under a 10× objective (Nikon E Plan LWD, 10×/0.25). We analysed MCA spontaneous myogenic tone generation and structural and mechanical properties. A 5 × 3 mm section of brain tissue containing the MCA was removed and placed in Ca2+‐free physiological salt solution (PSS) at 4°C, with 1% BSA. A branch‐free segment of the MCA was mounted on two glass micropipettes in the pressure myograph. MCAs were equilibrated at 80 mmHg in PSS containing (in mmol) 141.9 NaCl, 4.7 KCl, 1.12 KH2PO4, 1.7 MgSO4.7H2O, 2.8 CaCl2, 10 Hepes, 5 dextrose and 0.5 EDTA (pH 7.4) until development and maintenance of at least 20% spontaneous myogenic tone, which was calculated using the following formula: %tone = [1−(active lumen diameter/passive lumen diameter)] × 100. The ability of the MCA to maintain a stable increase in external diameter with each pressure increment, and the  5 generation of 20% tone ascertains the absence of leaks. Passive structure was assessed in Ca2+‐free PSS containing 0.002 m EGTA and intraluminal pressure was increased from 3 to 180 mmHg in 20 mmHg increments. Wall‐to‐lumen ratio and circumferential wall stress (Baumbach & Hajdu, 1993) as well as passive distensibility were calculated as described previously (Chan et al. 2010). The elastic modulus (β‐coefficient) was calculated from the stress/strain curves using an exponential model (y = ae β x), where β is the slope of the curve and is directly correlated to vascular stiffness. Reactivity of the MCA Endothelial function in the MCA was assessed by extraluminal perfusion of carbachol in a pressure myograph as per convention and previously described (Brueggemann et al. 2009; Wang et al. 2012). The MCA was then bathed in PSS pressurized at 80 mmHg to generate spontaneous myogenic tone. Increasing concentrations of carbachol (10−10 to 10−5 m) were then added to the bath. Carbachol, along with being a potent agonist of endothelial muscarinic receptors expressed in the MCA, cannot be degraded by acetylcholinesterase (Dauphin & Hamel, 1990). Furthermore, the low molecular weight of carbachol (182.696 g mol−1) favours a high capacity for migration through smooth muscle to activate the endothelial cells. After a thorough wash with calcium PSS, a single dose of 5‐HT (serotonin; H9523; Sigma, St Loius, MO, USA) was added (10−5 m) to the bath. 1.2.3 Immunohistochemical analyses Tissue collection and immunofluorescence Animals were sacrificed with an overdose of chloral hydrate (1 g kg−1, i.p.) and the MCA was collected and post‐fixed in 4% paraformaldehyde (PF), and then stored in 20% sucrose. A segment of the MCA from each animal of different experimental groups was embedded in optimal cutting temperature (OCT) freezing medium. Transverse 10 μm sections of the MCAs were cut on a cryostat microtome, collected in Superfrost Plus slides and stored at −80°C until further processing. The morphological assessment of MCAs was determined  6 by immunofluorescence using the primary antibodies: mouse α TGFβ1 (1:200, Abcam, Cambridge, MA, USA; ab64715), rabbit α collagen (1:1000, Abcam, ab292), mouse α collagen 3 (1:300, Abcam, ab6310), mouse α angiotensin II receptor 1 (AT1; 1:200, Abcam, ab9391), rabbit α angiotensin II receptor 2 (AT2; 1:600, Abcam, ab19134), rabbit α elastin (1:400, Millipore, Billerica, MA, USA, AB2039) and rabbit α smooth muscle actin (SMA) (1:400, Abcam, ab5694). The MCA sections were thoroughly washed in PBS and 0.1 m PBS containing 0.3% Triton X‐100 (PBST), followed by 1 h of incubation in 10% normal donkey serum in PBST. Sections were then incubated with the primary antibodies for 24 h at room temperature. Subsequently, sections were washed in PBST and incubated accordingly with Alexa‐fluor 488 donkey anti‐mouse (1:1000; Molecular Probes, Carlsbad, CA, USA) or donkey anti‐rabbit conjugated Cy3 (1:1000; Jackson Immuno Research, West Grove, PA, USA) for 1 h. Finally, all the sections were washed, mounted with Prolong Gold mounting medium (Molecular Probes) and observed in an AxioImager.M2 Zeiss microscope (HR R3; objective magnification 20×, ocular magnification 10×) using Zen 2 Pro. Objective: Zeiss PLAN NEOFLUAR 20×/0.50. Cy3, excitation peak = 550 μm, emission peak = 570 μm, tetramethylrhodamine filter settings. Alex Fluor 488 excitation peak = 495 μm, emission peak = 519 μm, fluorescein isothiocyanate filter settings. Resolution of collected images: 942,490,000 pixels cm–2 (i.e. 3.070 pixels μm–1). Quantification The intensity of immunoreactivity of morphological markers of the MCAs was assessed using Fiji Software (based on ImageJ, http://fiji.sc/Downloads#Fiji). For all primary antibodies except tyrosine hydroxylase (TH), staining intensity was averaged from four sections per animal selected from the middle third of the MCA. We generated a region of interest using the advential border of the artery and the lumenal interface as the outer and inner boundaries. Within this region of interest, relative intensity was quantified. We were careful to obviate the surrounding  7 connective tissue from within the region of interest. In all cases, a reference intensity of unstained tissue was measured to determine background intensity, which was deducted from the average intensity of each section to calculate the mean net staining intensity. Furthermore, a negative control (i.e. only secondary antibody added in the absence of primary antibody) was required for each protocol to ensure specificity of secondary to primary antibody (Frias et al. 2015). In addition, the area of the region of interest was normalized in every tissue section. Staining intensities determined in Sham were used as standardizing controls. 1.2.4 Immunohistochemical morphometric assessment of the MCA Using the SMA staining of the MCA, arterial wall thickness and lumen diameter were measured using Image J. For each section of an artery, four sites were selected representing each quadrant of the vessel, and a line was drawn across the span of staining perpendicular to the tangent line, and the length of this line was measured. The average of the four measurements was used to denote wall thickness for each section. Lumen diameter was measured by taking the average of the widest and the narrowest part of the lumen. Four sections were measured and averaged from each animal. Considering the emission peak of the secondary antibody (Cy3) used with the SMA primary antibody and numerical aperture of the microscope, our resolution limit for this assessment was 570 μm, and data were reported to the nearest whole number. 1.2.5 TH analysis of MCA whole mounts  A small segment of the MCA vessel was used to determine TH expression (1:1000; anti‐rabbit; Millipore; ref.: AB152, USA) by immunofluorescence using Cy3 secondary antibody, and therefore TH+ fibre expression. For this, the vessel was placed in a small Eppendorf tube and the immunoreaction proceeded as described above. Finally, the vessel was placed in a glass microscope slide, mounted with Prolong Gold mounting medium (Molecular Probes) and viewed in the microscope.  8 Analysis of TH innervation in MCA whole mounts Quantification was carried out manually on Z‐stack images from the MCAs of each animal using Fiji Software. A rectangular grid was divided into equal squares and was placed over the region of interest on each of the upper and lower planes of the vessel. A grid was superimposed over the entire imaging frame (44 μm2 per grid sector). From the grid, a 5 × 5 sector region of interest was selected (220 μm2), with the inclusion criteria that the entire region of interest is within the boundary of the artery. The region of grid and region of interest were chosen randomly between animals. The quantification of the fluorescence intensity and innervation density was conducted within this region. A measurement was obtained for longitudinal and circumferential density by counting the number of horizontal and vertical grid line crosses, respectively. A value for total innervation density (longitudinal + circumferential) was then calculated for the vessel region and compared across treatments (Hesp et al. 2012). 1.2.6 Statistical analyses  Body mass, brain mass, brain mass/body mass, spontaneous myogenic tone generation, 5‐HT responsiveness, passive structure at 80 mmHg and immunohistochemistry data were analysed by Student's t test or a non‐parametric alternative when the data did not fit a normal distribution model. Repeated measures ANOVA was also used for passive structure over increasing transmural pressures using pressure myography (time × group). Concentration–response curves for carbachol were further analysed by non‐linear regression (Hill equation) to calculate logEC50 and logECmax. All statistical analyses were carried out using GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA). Differences between means were considered statistically significant at P < 0.05. Data are shown as mean ± SEM.  9 1.3 Results 1.3.1 Physiological parameters At the end point of the experimental protocol, body mass (T3‐SCI: 315 ± 11 vs. Sham: 344 ± 5 g: P = 0.06), brain mass (T3‐SCI: 2.2 ± 0.04 vs. Sham: 2.2 ± 0.03 g: P = 0.85) and brain mass/body mass ratio (Sham: 0.63 ± 0.01 vs. T3‐SCI: 0.69 ± 0.03; P = 0.20) were not different between groups. 1.3.2 SCI detrimentally altered structure of the MCA After T3‐SCI, pressure myography demonstrated that animals experienced 10% reduced MCA lumen diameter (P < 0.05, Figure 1A and 1B). The MCA lumen cross‐sectional area was also reduced by 20% after T3‐SCI (P < 0.05), without concurrent changes in wall thickness or wall cross‐sectional area (Table 1), leading to a 14% greater wall‐to‐lumen ratio after T3‐SCI (P < 0.05, Table 1). Morphometrics ascertained using immunohistochemistry also showed reduced lumen diameter, and increased wall‐to‐lumen ratio after T3‐SCI, although this technique also demonstrated a 42% increased MCA wall thickness in the T3‐SCI cohort (P < 0.05, Figure 2). Stiffness was also increased substantially after T3‐SCI. Specifically, distensibility was reduced 40% after T3‐SCI (P < 0.05, Figure 3). Furthermore, a 40% reduction in vessel strain and a 10% reduction in vessel stress (both P < 0.05; Table 1) led to a 36% increase in β stiffness trending towards significance (P = 0.09, Figure 3)   10    Figure 1-1. Complete T3‐SCI led to inward remodelling of the MCA Pressure myography demonstrated complete T3‐SCI (n = 7) led to inward remodelling of the MCA (D), characterized by a decrease in lumen size (A and B), but no corresponding increase in wall thickness was detected using repeated measures (C). Comparisons were made using only physiological intraluminal pressures (40–180 mmHg). *Significantly different from Sham (n = 5; P < 0.05). Repeated measures ANOVA (main effect)  11    Figure 1-2. T3‐SCI results in hypertrophic inward remodelling of the MCA Morphometric analysis of smooth muscle actin immunohistochemical staining demonstrated evidence that T3‐SCI results in hypertrophic inward remodelling of the MCA, as evidenced by greater wall thickness (A), reduced lumen diameter (B) and increased wall‐to‐lumen ratio (C) in the T3‐SCI group. *Significantly different from Sham (P < 0.05). Unpaired t test.        12     Figure 1-3. Mechanical properties of the MCA after T3‐SCI A–C, mechanical properties of the MCA were deleteriously influenced by T3 complete SCI (n = 7) resulting in reduced distensibility (A), impaired stress–strain relationship (B) and a trend for increased β‐stiffness (C). D, MCA constrictive responses to 10−5 m 5‐HT. Spinal cord transection at the T3 level (n = 7) led to a trend indicating impaired vascular responsiveness compared to Sham‐injured animals (Sham; n = 5). *Significantly different from Sham (n = 5; P < 0.05). A and B: repeated measures ANOVA (main effect). C and D: unpaired t test.    13  Table 1-1. Ex vivo pressure myography data showing remodelling of the MCA after T3 spinal cord injury  Sham SCI Lumen diameter (µm) 297.0 ± 10.4 265.3 ± 5.3* Outer diameter (µm) 331.0 ± 11.4 299.4 ± 5*  Wall thickness (µm) 17.00 ± 0.61 17.07 ± 0. 5 Wall to lumen ratio  0.057 ± 0.001 0.065 ± 0.001*  Lumen CSA (µm2) 69620 ± 4867 55410 ± 2217*  Vessel CSA (µm2) 86460 ± 5952 71660 ± 2768*  Wall CSA (µm2) 16840 ± 1141 16250 ± 1016 Vessel strain 0.7333 ± 0.15 0.4400 ± 0.05*  Vessel stress 699.5 ± 14.97 625.1 ± 24.92* Distensibility  73.3 ± 14.7 44.0 ± 5.2 *  Values are mean ± SEM at an intraluminal pressure of 80 mmHg. CSA, cross-sectional area. *Significantly different from Sham (p< 0.05).    14 SCI did not change myogenic reactivity of the MCA but reduced MCA contractility Spontaneously generated myogenic tone was not different between groups in absolute terms (Baseline: Sham = 308.2 ± 12.3 vs. T3‐SCI = 280.2 ± 22.4 μm; Post‐Myogenic Tone: Sham = 199 ± 36 vs. T3‐SCI = 184 ± 32 μm; P = 0.86), or as a per cent (Sham = 36 ± 4; T3‐SCI = 35 ± 3%; P = 0.87). We did observe a trend for a reduced contractile response to 10−5 m 5‐HT which was on average 33% lower in T3‐SCI (P = 0.09; Figure. 3d) SCI did not impair endothelial function in the MCA After T3‐SCI, there was no difference in MCA logEC50 or logECmax (Figure 4). Specifically, endothelial function of the MCA at 80 mmHg was not significantly different between groups. Neither maximal dilatation to carbachol (logECmax) or sensitivity (logEC50) was different between Sham and T3‐SCI SCI‐mediated profibrosis within the MCA After T3‐SCI, there was 42% increase in collagen I and a 24% increase in collagen III expression within the MCA coinciding with a 47% increase in TGF‐β (P = 0.04, P = 0.005 and P = 0.02, respectively; Fig. Fig.5).5). Elastin was reduced 27%, while angiotensin II receptor type 2 was increased 132% (P = 0.04 and P < 0.001, respectively; Fig. 5). Figure 5 C shows for representative staining for each primary antibody.    15    Figure 1-4. Endothelial function of the MCA after SCI  Endothelial function of the MCA, assessed at 80 mmHg with carbachol, was not significantly impacted by SCI. Specifically, neither maximal dilatation to carbachol (logECmax) nor sensitivity (logEC50) was different between Sham (n = 5) and T3 complete SCI animals (n = 6). One‐way ANOVA.  16   Figure 1-5. Profibrotic middle cerebral artery after SCI A, immunofluorescence intensity of proteins associated with profibrosis between Sham and T3‐SCI. Values are mean ± SEM. TGF‐β, transforming growth factor beta; AT1, angiotensin II receptor type 1; AT2 angiotensin II receptor type 2; SMA, smooth muscle actin. *Significantly different from Sham (P < 0.05). Unpaired t test. B, selection of the region of interest (ROI) for quantification of staining intensity used the adventitial layer as the outer boundary and the medial border of the internal elastic lamina (IEL) as the inner boundary. C, example immunohistochemical images at 20× magnification from Sham (middle) and SCI animals (right) for: CI, collagen 1; CIII, collagen 3; TGFβ, transforming growth factor beta. Inset: example staining for each primary antibody at 63× magnification.  17  Complete SCI at the T3 level did not reduce sympathetic innervation of the MCA After the present model of T3 complete T3‐SCI, no reduction in TH+ axonal density or fluorescence intensity occurred (Figure 6).   18   Figure 1-6. TH+ axonal density and fluorescence intensity after T3‐SCI Top: representative immunohistochemical images from Sham (left; n = 3) and T3‐SCI animals (right; n = 8) for tyrosine hydroxylase (TH). Bottom left: TH immunoreactive (TH‐ir) relative axon density of the MCA. Bottom right: white arrows denote which axons were considered TH positive. No differences occurred between Sham injured and animals with T3 complete spinal cord injury. Unpaired t test.  19 1.4 Discussion This is the first study indicating that high‐thoracic SCI leads to extensive cerebrovascular maladaptation, including hypertrophic inward remodelling, stiffening and possibly impaired reactivity. We also demonstrate that the cerebrovascular environment is profibrotic after high‐thoracic SCI, characterized by increased collagen, decreased elastin and increased expression of TGF‐β, a protein involved in the signalling cascade leading to fibrosis. As sympathetic pathways to the brain vasculature were not disrupted in the present T3 transection model, it is our contention that these deleterious alterations in the cerebrovasculature are a secondary consequence of a combination of lower capacity for physical exercise, reduced alterations in blood volume/pressure after T3‐SCI and an over‐reliance on the RAS (Noreau et al. 1993; Wecht et al. 2009). These factors have each been shown to induce profibrosis (Leask & Abraham, 2004; Duprez, 2006; Marchesi et al. 2008; Tuday et al. 2009; Sofronova et al. 2015). The present findings provide evidence demonstrating advanced arteriosclerotic progression in the brain after T3‐SCI and insight into the underlying mechanisms. Taken together, a profibrotic environment within the cerebrovasculature after T3‐SCI probably plays a role in a variety of cerebrovascular dysfunctions after SCI. These include the increased risk of stroke, as well as other cerebrovascular conditions such as dysfunctional cerebral blood flow regulation and vascular cognitive impairments (Cragg et al. 2013; Wiesmann et al. 2013; Phillips et al. 2014 a,b), and as well may lead to reduced global cerebral blood flow (Phillips et al. 2013 a). Remodelling of the MCA has been well documented in a variety of clinical conditions (Pires et al. 2013; Dorrance et al. 2014). After T3‐SCI, the present study illustrated a decrease in MCA lumen diameter, increased wall thickness and a significant increase in wall‐to‐lumen ratio. Increased MCA stiffness in the present T3‐SCI cohort demonstrates that cerebrovascular  20 remodelling after T3‐SCI is probably directly impacting the distensibility of this vessel. Mechanistically, the reduced distensibility would be expected considering the corresponding immunohistochemical assessments demonstrating increased collagen and decreased elastin expression in the MCA after T3‐SCI (Izzard et al. 2006). Previous research has implicated the loss of suprapinal sympathetic tone as a mechanism leading to arterial remodelling. Indeed, in a rabbit model, increased collagen and decreased elastin were shown in the aorta after chronic sympathectomy (Fronek et al. 1978). It is unlikely we disrupted descending sympathetic pathways of supraspinal origin in the present study, however, as we have shown that sympathetic fibre density of the MCA is identical between the two groups, and would have been decreased or absent if the sympathetic pathway was disrupted (Hesp et al. 2012). There are reductions in physical activity and blood volume/pressure after cervical and high‐thoracic SCI (Scott et al. 2011), and we have previously reported that blood pressure is significantly reduced following T3 spinal cord transection in Wistar rats (West et al. 2015). The combination of these factors probably exacerbates large artery dysfunction in our model of T3 complete SCI, as reduced physical activity and blood volume/pressure have both been implicated in the noted large artery dysfunction (Geary et al. 1998; Zhang et al. 2001; Tuday et al. 2009). These studies reported similar but not identical findings to the present study, where profibrosis occurred and vasoreactivity of the cerebrovasculature was impaired after exposure to reductions in physical activity, and reduced blood volume. It is interesting to note that one of the mentioned studies showed a counterintuitive increase in vasoreactivity of the basilar artery, although this may be due to regional vasoreactivity differences within the cerebrovasculature as has been previously reported (Sato et al. 2012; Phillips et al. 2014 a).  21 In addition to reduced exercise capacity and autonomic instability, after cervical and high‐thoracic SCI the cardiovascular system also adapts to rely predominantly on the RAS for blood pressure maintenance (Wecht et al. 2005; Handrakis et al. 2009; Groothuis & Thijssen, 2010). Increased RAS leads to a profibrotic environment, which is characterized by excessive production, deposition and contraction of the extracellular matrix (Leask & Abraham, 2004). The protein TGF‐β is a major profibrotic signalling cytokine that is upregulated by elevated RAS activity (Rosendorff, 1996). As evidenced by our T3‐SCI animals, SCI results in elevated collagen content and increased TGF‐β. It is worth noting that TGF‐β is typically considered an up‐regulator of elastin expression, although increases in RAS activity have also been shown to lead to elastin degradation (Pons et al. 2011). It appears in the present study that the latter elastin degradation trumped the upregulation expected from increased TGF‐β expression. Also, only AT2 receptors were increased after T3‐SCI in the present study, although this may have occurred as an adaptation to counteract the profibrotic status induced by increased RAS activation of the AT1 (Montezano et al. 2014). Accordingly, it is fair to posit that after T3‐SCI profibrosis develops within the large cerebral arteries (probably due to increased RAS activity) leading to overexpression and contraction of the extracellular matrix, resulting in reduced distensibility and hypertrophic remodelling. After T3‐SCI, reduced lumen diameter, increased stiffness and ensuing increased vascular resistance of the MCA likely leads to the frequent observation of reduced cerebral blood flow (Phillips et al. 2013 a), which would exacerbate disorders associated with cerebral hypoperfusion, including vascular dementia, Alzheimer's disease and recovery from ischaemic stroke (Vorstrup et al. 1986; Farkas & Luiten, 2001; Roher et al. 2012). Furthermore, increased fibrosis within the cerebrovasculature is probably contributing to dysfunctional cerebrovascular  22 regulation after cervical and high‐thoracic SCI (e.g. impaired neurovascular coupling, cerebrovascular reactivity) (Wilson et al. 2010; Phillips et al. 2013 b). Endothelial function of the MCA was not diminished after T3‐SCI, which is in agreement with several studies showing normal endothelial function in vasculature rostral to the lesion level after SCI (de Groot et al. 2004, 2006). Considering the already elevated risk of stroke in this population, any decrease in endothelial function would further exacerbate this risk by inhibiting the capacity to circumvent occlusion through collateral artery dilatation (Coyle, 1987) There was a trend suggesting a reduction in vascular reactivity to 5‐HT after T3‐SCI in the present study. Although this could be attributed to increased profibrosis and stiffness of the MCA, the fact that endothelial responsiveness was not also impaired suggests that factors other than increased stiffness of the MCA could be responsible. One possibility is that an increase in sympathetic nervous system activity above the lesion level (i.e. from the T1–2 levels) after T3 complete SCI impacted reactivity. It well documented that sympathetic nervous system activity is inversely associated with vascular constrictive sensitivity (Brock et al. 2006; Charkoudian et al. 2006), and we have shown that after high‐thoracic SCI, there are indications of increased sympathetic activity to target organs above the lesion level (Claydon & Krassioukov, 2008; Lujan et al. 2010, 2012), which would be expected to lead to vasoconstrictor hyposensitivity rostral to injury. From a clinical perspective, impaired vasoconstrictor function of the MCA would certainly inhibit functional cerebral autoregulation, particularly during periods of hypertension, such as autonomic dysreflexia, which are well reported to lead to cerebral haemorrhage (Wan & Krassioukov, 2014).  There are a number of exciting directions for future research, after illustrating for the first time impaired cerebrovascular structure and function after experimental T3‐SCI. First, the  23 mechanism underlying the profibrotic environment in the vasculature after T3‐SCI needs to be elucidated. An initial step would be to assess the effect of AT1/AT2 receptor blockade/exercise training after high‐thoracic SCI, with a particular focus on matrix metalloproteinases known to impact collagen and/or elastin (Zieman et al. 2005). Another necessary step is to understand the role autonomic dysreflexia (i.e. transient hypertensive episodes that occur on a daily basis after high‐thoracic or cervical SCI) plays in cerebrovascular health in this population (Hubli et al. 2015). Moreover, understanding how profibrotic cerebrovascular remodelling affects cerebral blood flow regulation in this model would be a logical extension of the present study. We did not assess behaviour outcomes related to cerebrovascular dysfunction secondary to T3‐SCI, such as cognitive function. It would be ideal to assess cognitive function after high‐thoracic SCI as compared to Sham. Given the significant motor dysfunctions of the hind limbs after SCI, however, it is difficult to truly assess cognitive function through standard protocols such as the Morris water maze. We are certain that dilatation after carbachol administration in the present study was due to endothelial activation, although it is possible that greater magnitude dilatation would have occurred if carbachol was administered intralumenally. It is also possible that greater between‐group differences would have occurred for 5‐HT if a range of doses were used. Different vessel preparations (i.e. ex vivo living tissue vs. post‐fixed frozen tissue) may have led to the discrepancy in absolute wall thickness between pressure myography and immunohistochemistry staining. Immunohistochemistry was used to indirectly quantify protein expression in the present study. Although clinically this technique is not considered quantitative due to a lack of reference standards, immunohistochemistry, when applied using identical filtering and staining protocols as well as ensuring a dark negative control, can provide accurate quantification of protein expression (Taylor & Levenson, 2006; Frias et al. 2015).  24 1.5 Conclusions Structure and function of the cerebrovasculature are deleteriously impacted by high‐thoracic or cervical SCI, secondary to the development of a profibrotic environment. This profibrosis may be the consequence of a combination of an increased influence of RAS, reduced physical activity and reductions in blood pressure/total blood volume in this model of SCI, but not the direct impact of decentralized sympathetic pathways to the brain. Starkly increased risk of stroke, dysfunctional cerebral blood flow regulation and vascular cognitive impairments may all be associated with impaired large cerebral artery structure and function after SCI as illustrated in the present study. Acknowledgements Paul Lesack (Data/GIS Analyst, University of British Columbia) is acknowledged for his remarkable artistic contributions and technical expertise in fulfiling our vision for the cover art associated with this manuscript: ac.cbu@kcasel.luap. Dr Matthew Ramer is acknowledged for his extensive consultation regarding immunohistochemistry protocols.  25 Chapter 2. Transient cerebral hyper-perfusion due to autonomic dysreflexia impairs cerebrovascular function and structure 2.1 Introduction Chronic steady-state hypertension is the most powerful modifiable risk factor for cerebrovascular diseases(O’Donnell et al. 2010) and, as such, has received appropriate clinical and research attention. As a clinical community, we have become aware more recently that in addition to chronic hypertension, there is also significantly elevated cerebrovascular disease risk in persons with chronic exposure to labile blood pressure, such as repetitive transient hypertension(Rothwell et al. 2010). Repetitive bouts of transient hypertension are common in a vast number of prevalent activities including acute caffeine consumption,3 resistance exercise,4 high-intensity interval exercise,5 defecation, exaggerated blood pressure responses to mental stress,6 and drug abuse.7 The majority of these stimuli have been linked with stroke6,8,9; however, the direct cerebrovascular consequences of transient hypertension have never been examined(Everson et al. 2001; Klonoff, Andrews, and Obana 1989; Lucas et al. 2015; MacDougall et al. 1985; Mostofsky et al. 2010; Resnick, Kestenbaum, and Schwartz 1977; Vlachopoulos et al. 2004).  Repetitive exposure to transient hypertension may exert a unique form of cerebrovascular decline compared with the well characterized pathophysiology associated with steady-state hypertension, which includes endothelial dysfunction, fibrotic hypertrophic inward remodeling, reduced cerebral blood, and elevated cerebrovascular myogenic tone(Baumbach and Heistad 1988; Gibbons and Dzau 1994). Understanding the structural and functional changes in the  26 cerebrovasculature resulting from transient hypertension would be invaluable to help understand and potentially manage this condition.  Spinal cord injury (SCI) provides a useful model to study transient hypertension, because injury above the sixth thoracic spinal segment leads to the loss of supraspinal control of autonomic spinal circuitry, resulting in vascular and neuroanatomical changes caudal to the injury, predisposing the spinal sympathetic circuitry to overreact to afferent stimuli, leading to rapid elevations in blood pressure. These episodes of sympathetic overactivation most often result from a full bladder/bowel or sexual stimulation after SCI. Blood pressure can elevate in a matter of seconds up to 300 mm Hg, a phenomena termed autonomic dysreflexia (AD)(Furlan et al. 2003; Hubli, Gee, and Krassioukov 2014). These transient increases, which occur on average 11 times/day after high-level SCI,12 can elicit serious clinical consequences. For example, we and others have shown that patients with SCI not only have a staggering 3-4–fold increased odds/risk of stroke (six strokes/1000 person-years), but also from significant cerebrovascular dysfunction(Cragg et al. 2013; Phillips et al. 2017; Phillips, Krassioukov, et al. 2014; Phillips, Warburton, et al. 2014; Wu et al. 2012).  Here, we first establish the clinical reality of transient hypertension in humans with SCI by characterizing the acute cerebrovascular responses to transient hypertension from AD. Next, to invasively examine the structural alterations and underlying pathophysiology resulting from transient hypertension, we developed a pre-clinical model of transient hypertension in SCI that mimics the clinical scenario. Following this, we employed the model to evaluate whether chronic (four weeks) transient hypertension impairs cerebrovascular function and structure using in vitro pressure myography of the middle cerebral artery as well as magnetic resonance imaging (MRI) to evaluate the in vivo impact on regional and global cerebral blood flow (CBF). Extrapolating  27 from the chronic steady-state literature, we hypothesized that chronic exposure to transient hypertension would result in declined cerebrovascular health evidenced by impaired endothelial function, profibrotic protein expression, altered constrictive responsiveness, and reduced global CBF(Pires et al. 2013; Yang et al. 1991).  2.2 Methods  Clinical evaluation We first evaluated the acute effects of transient hypertension on CBF during 12 episodes of AD occurring in six patients undergoing iatrogenic procedures known to elicit AD (i.e., urodynamic filling and penile vibrostimulation for sperm retrieval)(Fougere et al. 2016; Phillips, Elliott, et al. 2014).  2.2.1 Experimental protocol.  Both urodynamic filling and penile vibrostimulation for sperm retrieval are clinical procedures required for persons with SCI, which unfortunately are well documented to elicit AD iatrogenically(Aaron A. Phillips et al. 2016; Phillips, Elliott, et al. 2014). Participants included eight male individuals (n = 3 urodynamics assessments, n = 5 penile vibrostimulation) with complete cervical chronic SCI (age 37 – 4 years; n = 4 American Spinal Injury Association Impairment Scale [AIS]-A; n = 2 AIS-B; n = 2 AIS-C; all >2 years post-SCI). The level and completeness of injury was determined by a licensed physician (AVK). Participants had no history of cardiovascular disease. Informed consent was obtained from all subjects, and the protocol was approved by the University of British Columbia Clinical Research Ethics Board and adhered to the Declaration of Helsinki.  For both urodynamics and penile vibrostimulation, blood pressure was recorded every minute on the right arm using an automated sphygmomanometer (DinamapV100; GE Medical Systems, Fairfield, CT), using a fitted cuff. Three supine measurements were taken before the procedure and averaged to determine baseline supine blood pressure. Beat-by-beat blood pressure  28 and CBF were measured as described previously(Aaron A. Phillips et al. 2016; Phillips, Warburton, et al. 2014). 2.2.2 Urodynamic assessments.  Before urodynamics, a urinary dipstick test confirmed the absence of infection. Urodynamics occurred between 0800 and 1200 h in a temperature-controlled room (21C) according to established standards(Biering-Sørensen et al. 2008). Cystometry was performed by a double-lumen catheter (6 Fr, Laborie, Canada) with continuous filling of sterile water (37C) at a fixed rate of 30 mL/ min. Abdominal pressure was measured with an intrarectal balloon catheter (10 Fr, Laborie, Canada). Pelvic floor electromyography (EMG) (Aquarius TT, Laborie Model 94-R03-BT, Montreal, Quebec, Canada) was recorded using surface EMG. Filling was stopped if any of the following conditions were observed: (1) reported sensation of fullness; (2) spontaneous urine leakage; (3) intravesical pressure >= 40 cm H2O; (4) infused volume reached 500 mL; or (5) sustained systolic blood pressure >= 180 mm Hg or intolerable AD symptoms (e.g., severe goose bumps, sweating, headache, perceived head pressure pulsations that were not present previously, etc.).  2.2.3 Penile vibrostimulation for sperm retrieval.  Penile vibrostimulation was applied by a physician experienced in penile vibrostimulation using one or more handheld vibrators (Ferti Care, Multicept, Rungsted, Denmark) placed about the glans penis to induce ejaculation. An amplitude of 1.0–3.5 mm with a frequency of 70–100 Hz was used. Vibration was performed for a maximum of 3 min, followed by a pause of 1 min before the cycle was repeated until ejaculation was achieved. 2.2.4 Clinical cardiovascular and cerebrovascular assessments.  The following were sampled at 1000 Hz using an analogto-digital converter (Powerlab/16SP ML 795; ADInstruments, Colorado Springs, CO) interfaced with data acquisition software (LabChart8;  29 ADInstruments): beat-by-beat blood pressure via finger photoplethysmography (Finometer PRO; Finapres Medicine Systems, Amsterdam, Netherlands), electrocardiogram (ML132; ADInstruments) on the left side, and blood velocity in the left middle cerebral artery (ST3 Transcranial Doppler, Spencer Technologies, Redmond, WA). The middle cerebral artery was insonated using a 2 MHz probe mounted on the temporal bone and a fitted headstrap. The middle cerebral artery (MCA) was insonated at a depth of 45–55 mm and confirmed using ipsilateral common carotid artery compression.  The autonomic dysreflexia was defined by a >= 20 mm Hg in increase in systolic blood pressure and confirmed by a physician with expertise in this condition (AVK). Mean arterial blood pressure was calculated as ([2*diastolic blood pressure] + [systolic blood pressure])/3 while mean CBF velocity was calculated by ([2*minimum flow velocity] + [peak flow velocity]) /3. Together a scientist and clinician with expertise in autonomic dysreflexia identified at least three heart beats corresponding to maximum blood pressure (typically three beats) during AD were extracted to calculate the peak change in blood pressure and CBF.  Pre-clinical model  2.2.5 Experimental protocol.  Surgery and animal care were conducted according to standard procedures in our laboratory to perform a complete spinal transection at the T3 spinal segment(A. A. Phillips et al. 2016). Four male rats (age = nine weeks, mass = 250–350 g; Harlan Laboratories, Indianapolis, IN) were used initially to develop the translational model of transient hypertension and ensuing cerebral hyperperfusion. Next, using this model, 50 male rats (identical age, strain, and supplier) were randomly assigned either (Fig. 1d): SCI (T3-SCI; n = 25), or SCI with AD (T3-SCI+AD; n = 25). Two weeks after SCI, repetitive AD induced by colorectal distension (explained below) was initiated for four weeks in the T3-SCI+AD group, which is analogous to two years of human  30 life(Phillips, Elliott, et al. 2014). Six weeks after SCI, both groups underwent MRI assessment and were then sacrificed for a range of vascular assessments spanning pressure myography, histology, and molecular biology. All outcome assessments and analysis were performed by blinded personnel.  2.2.6 Pre-clinical spinal cord transection.  Animals received prophylactic enrofloxacin (Baytril; 10 mg kg-1, subcutaneously [SC], Associated Veterinary Purchasing [AVP], Langley, Canada) for three days before the SCI surgical procedure. On the day of operation, enrofloxacin (10 mg kg-1, SC), buprenorphine (Temgesic; 0.02 mg kg-1, SC, McGill University), and ketoprofen (Anafen, 5 mg kg-1, SC, AVP) were administered pre-operatively. Rats were anesthetized with ketamine hydrochloride (70 mg kg-1, intraperitoneally [IP]; Vetalar; AVP) and dexmedetomidine (0.25 mg kg-1, IP; Domitor; AVP).  A dorsal midline incision was made in the superficial muscle overlying the C8–T3 vertebrae. The dura was opened at the T2–T3 intervertebral gap, and the spinal cord was completely transected using microscissors. Complete transection was confirmed by two surgeons via visual separation of the rostral and caudal spinal cord stumps, and Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York) was placed between the stumps to achieve hemostasis. The muscle and skin were closed with 4–0 Vicryl and 4–0 Prolene sutures, respectively.  Animals received warmed lactated Ringer solution (5 mL, SC) and recovered in a temperature-controlled environment (Animal Intensive Care Unit, Los Angeles, CA, USA), after being administered atipamezole (1mg kg-1, SC, AVP) for reversal of dexmedetomidine. Enrofloxacin (10 mg kg-1, SC), buprenorphine (0.02 mg kg-1, SC) and ketoprofen (5 mg kg-1, SC) were administered once a day for three days post-operatively. The bladder was expressed manually three to four times daily until spontaneous voiding returned (about 10 days post-injury). All assessments and analyses were completed blinded of each condition/group using a secret animal  31 coding system known only to the project lead. Five animals did not survive the SCI transection procedure. Male animals were used because they represent the majority of SCI(Phillips and Krassioukov 2015). 2.2.7 Ultrasound evaluation of middle cerebral artery blood flow with concurrent blood pressure during acute AD/transient hypertension induced by colorectal distension.  After being anesthetized with urethane (1.5–2.5 g/kg), a calibrated blood pressure catheter (SPR-869, Millar), was implanted in the right femoral artery and was advanced into the right common iliac artery. Further, a 20 mm· 10 mm section of the crown of the skull was removed, with care to not pierce the dura. Blood pressure recordings were collected and recorded concurrent to the ultrasound recordings by being input through analog to the ultrasound device (Vevo 3100; Visual Sonics, Inc., a subsidiary of Sonosite, Bothell, WA).  A balloon tip of a Swan-Ganz catheter (10 mm in length) was inserted 2 cm rectally, according to standard procedures to induce transient hypertension secondary to AD.27 For this, the balloon was infused with 1.0–2.0 mL of air for 60 sec. Three uninjured rats of the same age had transient hypertension induced by bolus injections of 0.05–0.2 mL (50 lg/mL) phenylephrine (because colorectal distension does not lead to transient hypertension in uninjured rats) through the tail vein, which was cannulated with a 26-gage butterfly needle.  During scanning, the rat was fixed to a customized scanning platform (Visual Sonics), which was heated to maintain body temperature, while the ultrasound probe (MX250, 24 MHz, Visual Sonics) was aligned to the frontal plane and moved rostrally and caudally; color Doppler was applied to aid in localization of the middle cerebral artery during B-mode imaging (inset Fig. 1b) as described previously.17 The ultrasound frequency was 16 MHz, while the pulsed repetition frequency was set to 4 kHz. The Doppler sample volume was placed at a depth of 8–11 mm, with Doppler angle set to less than 60 degrees. If we deviated from this for optimization, then these  32 settings were kept consistent throughout the hyperemia. Baseline values and maximum increase in CBF were extracted, along with the corresponding blood pressures. Mean arterial pressure was calculated by = ([2*diastolic blood pressure] + [systolic blood pressure])/3 while mean CBF velocity was calculated by = ([2*minimum flow velocity] + [peak flow velocity])/3.  2.2.8 Repetitive colorectal distension to induce AD.  At 14 days post-SCI, the T3-SCI+AD group underwent six successive bouts of AD that we have demonstrated leads to transient hypertension, five days/week for four weeks, each of 10 min duration. In short, the balloon tip catheter was inserted identically as described above; however, in this case inflation was maintained for 10 min(West et al. 2014).  Pressure myography. Seven weeks after injury, T3-SCI and T3-SCI+AD rats were anesthetized with 3% isoflurane and euthanized by decapitation after exsanguinations for ex vivo pressure myography studies. The MCA function and structure were assessed by pressure myography (Living Systems, St. Albans, VT) as described previously(A. A. Phillips et al. 2016). Briefly, we analyzed MCA spontaneous myogenic tone generation and structural and mechanical properties.  A 5 x 3 mm section of brain tissue containing the MCA was removed and placed in Ca2+ -free physiological salt solution (PSS) at 4C, with 1% bovine serum albumin. A branch-free segment of the MCA was mounted on two glass micropipettes in a pressure myograph (Danish Myo Technology, Aarhus, Denmark). The MCAs were equilibrated at 80 mm Hg in PSS containing (in mmol) 141.9 NaCl, 4.7 KCl, 1.12 KH2PO4, 1.7 MgSO4·7H2O, 2.8 CaCl2, 10 HEPES, 5 dextrose, and 0.5 EDTA (pH 7.4) until development of spontaneous myogenic tone, which was calculated using the following formula: %tone = (1 - [active lumen diameter/passive lumen diameter]) · 100. Passive structure was assessed in calciumfree PSS containing 2 mmol/L EGTA, and intraluminal pressure was increased from 3 to 180 mmHg in 20-mm Hg increments. The wall-to-lumen ratio and circumferential wall stress were calculated(A. A. Phillips et al. 2016).  33 Passive distensibility was calculated as described previously(A. A. Phillips et al. 2016). The elastic modulus (b-coefficient) was calculated from the stress/strain curves using an exponential model (y = aebx), where b is the slope of the curve and is directly correlated to vascular stiffness.  2.2.9 Reactivity of the MCA.  Endothelial function in the MCA was assessed by extraluminal perfusion of carbachol in a pressure myograph. A branch-free segment of the MCA was isolated and mounted between two glass cannulas in a pressure myograph chamber, which was then placed in an inverted microscope coupled to a camera. The MCA was then bathed in PSS, pressurized at 80 mm Hg to generate spontaneous myogenic tone. Increasing concentrations of carbachol (10-10 M to 10-5 M) were then added to the bath. After thorough wash with calcium PSS, a single dose of 5- HT (Sigma: H9523) was added (10-5 M) to the bath. Changes in MCA diameter were recorded using the MyoView software (Danish Myo Technology, Aarhus, Denmark).  2.2.10 Immunohistochemical analyses.  Briefly, the MCAs sections were thoroughly washed in phosphate buffer saline (PBS) and 0.01 M PBS containing 0.3% Triton X-100 (PBST), followed by a 1 h incubation in 10% normal donkey serum in PBST. Sections were then incubated with the primary antibodies listed in Table 1 overnight at room temperature. Subsequently, sections were washed in PBST and incubated accordingly with Alexa-fluor 488 donkey [I_AAP1] anti-mouse (1:1000; Molecular Probes ©) or Alexa Fluor 594-conjugated donkey anti-rabbit (1:1000; Jackson ImmunoResearch) for 1 h. Finally, all the sections were washed, mounted with Vectashield mounting medium with DAPI (Vector Laboratories Inc.) and observed in laser confocal AxioImager.Z2 Zeiss© LSM 800 microscope using identical exposure times, scanning speeds, gains, and pinhole values. The intensity of immunoreactivity of morphological assessment of MCAs was assessed using the Fiji Software (based on ImageJ, http://fiji.sc/ Downloads#Fiji).  34 2.2.11 Tyrosine hydroxylase (TH) analysis of MCAs whole mounts.  A small segment of the MCA vessel was used to determine TH expression (1:1000; anti-rabbit; Millipore; ref: AB152) by immunofluorescence. For this, the vessel was placed in a small Eppendorf tube and the immunoreaction proceeded as described above. Finally, the vessel was placed in a glass microscope slide, mounted with Prolong Gold© mounting medium (Molecular Probes©) and observed in the confocal microscope. The intensity of immunoreactivity of morphological assessment of MCAs was assessed using the Fiji Software (based on ImageJ). Staining intensity for each animal was determined from the middle third of the MCA.  2.2.12 Morphological assessment of MCA.  A segment of the MCA from each animal of different experimental groups was embedded in optimal cutting temperature (OCT) freezing medium. Transverse 10 lm sections were cut on a cryostat microtome, collected in Superfrost Plus slides, and stored at -80C until further processing. The morphological assessment of MCAs was determined by immunofluorescence using the primary antibodies listed in Table 1.  2.2.13 Molecular biology assessment of the MCA.  Total RNA was extracted from the MCA using the ribonucleic acid (RNA) Micro Kit (Purelink; Carlsbad, CA) following the manufacturer’s instructions. For the MCA tissue, the Rotor-Stator Protocol was used, and the TRIzol® Reagent Protocol was used for the aorta tissue. The concentration and purity of the extracted RNA were checked by measuring the optical densities at 260 and 280 nm. Total RNA (2.5 lg) was reverse transcribed into cDNA using InvitrogenTM SuperScriptTM VILOTM mastermix (Invitrogen; Carlsbad, CA), and following the manufacturer’s guidelines. Template cDNA was mixed with this mastermix, and the expression of genes involved in proinflammatory and profibrosis was studied using 96- well PCR array plates (MicroAMP® Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL), 4346906, Applied  35 Biosystems). Quantitative PCR (qPCR) was performed on an Applied Biosystems ViiATM 7 Real-Time PCR System machine using the following PCR cycling conditions: 50C for 2 min, and 95C for 10 min, and 40 cycles at 95C for 15 sec and 50C for 1 min. The gene expression fold change relative to the control was calculated using the 2-ΔΔCT method. 2.2.14 CBF  MRI was used to estimate CBF with the arterial spin labeling (ASL) technique. In brief, ASL tags the magnetization of inflowing blood by spin inversion, which modifies the overall signal of a downstream brain slice by an amount proportional to the amount of incoming labeled blood. The resultant CBF describes the amount of arterial blood delivered to the imaging slice within the transit time of the experiment. Experiments were performed on a preclinical 7 Tesla MRI scanner (Bruker Biospec 70/30 USR, Bruker, Ettlingen, Germany) using a quadrature volume transmit coil and four-channel receive phased-array. Body temperature was maintained by heated air and rectal temperature probe. The animal was maintained under 2% isoflurane anesthesia on a ventilator (Harvard Apparatus Inspira ASV) at 60–80 breaths per minute with expired CO2 measured with a Physiosuite capnograph (Kent Scientific) and respiration rate measured by a pressure sensor on the chest.  Multiple inversion recovery echo-planar imaging (EPI) scans were acquired to provide calibration data (tissue T1, inversion efficiency, and proton density) for ASL scans (recovery time = 10 sec, echo time (TE) = 11.47 ms, 22 inversion times between 30–8000 ms spaced geometrically, field of view = 40 · 40 mm, matrix = 64 · 64, slice thickness = 2 mm, nine slices, single shot). FAIR-EPI with a pre-saturation preparation was used to acquire the ASL data at three inversion times to allow correction for variable transit times (inversion times = 600, 800, 1000 ms, single-shot readout, TE= 10.5 ms, recovery time after pre-saturation = 1.1 sec, same geometry as calibration scans, GRAPPA factor = 2, partial-FT acceleration = 1.5, imaging time = 10 min 6 sec).  36 2.2.15 Statistical analyses.  The relationship between blood pressure and CBF in humans and rats was evaluated using the Pearson linear correlation. Spontaneous myogenic tone generation, 5-HT constriction, passive structure at 80 mm Hg, and immunohistochemistry data were analyzed by the Student t test or a nonparametric alternative. Concentration-response curves for carbachol were analyzed further by nonlinear regression (Hill equation) to calculate logEC50 and logECmax. All statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad, San Diego, CA). The difference between means was considered statistically significant when p < 0.05. Data are shown as mean– standard error of the mean.  2.3 Results  2.3.1 Clinical evaluation during transient hypertension.  The average resting supine baseline blood pressure for all episodes was 126– 5 mm Hg. The elevations in mean arterial pressure (penile vibrostimulation = 43– 5 vs. urodynamic filling = 46– 6 mm Hg; p = 0.77) and CBF (penile vibrostimulation = 21 – 4 vs. urodynamic filling = 23 – 4 cm/sec; p = 0.38) were not different between penile vibrostimulation and urodynamics. We observed that increases in brain perfusion pressure (i.e., blood pressure) because of transient hypertension are not buffered by cerebral autoregulation (i.e., the capacity to regulate and maintain CBF constant in the face of changing blood pressure), but instead lead to cerebral hyperperfusion, as evidenced by 2-3–fold increases in CBF (+23 – 4 cm/sec; Fig. 1a), and a strong significant positive correlation between increasing blood pressure and CBF (R2 = 0.76, p < 0.0001).  2.3.2 Pre-clinical model of transient hypertension  Our clinically relevant model elicits similar relative increases in CBF during autonomic dysreflexia as those observed during iatrogenic procedures in humans (+22 – 3 mm/sec; see Fig. 1b), and resulted in a similarly and significant positive correlation between increasing blood  37 pressure and CBF (R2 = 0.65, p = 0.001). Further, uninjured animals had very similar blood pressure and CBF responses to transient hypertension secondary to phenylephrine (R2 = 0.61, p = 0.01; Fig. 1c).  2.2.4 Cerebrovascular consequences of chronic transient hypertension  Our data demonstrate that repetitive exposure to cerebral hyperperfusion secondary to transient hypertension dramatically impairs endothelial function (Fig. 2a). Specifically, chronic exposure to repetitive autonomic dysreflexia led to reduced maximal dilation to carbachol (logECmax; but not sensitivity [logEC50]) (Fig. 2a). Chronic exposure to repetitive autonomic dysreflexia also resulted in stiffening the middle cerebral artery, such that distensibility was reduced (Fig. 2d), while strain (Fig. 2c) and beta stiffness (Fig. 2d) also trended to be impaired compared with animals with SCI. Chronic exposure to repetitive autonomic dysreflexia did not lead to remodeling, such that lumen diameter, wall thickness, and wallto-lumen ratio were similar between groups (Fig. 2c); however, the middle cerebral artery constricted 37% more in response to 10-5M serotonin (5-HT) (Fig. 2e). Percent tone generation of the middle cerebral artery was not different between groups at 80 mm Hg (28.5 – 2.1% vs. 31.4 – 2.1%). These changes were accompanied by cerebrovascular profibrotic expression characterized by increased expression of collagen (Fig. 3a).  These findings, along with our in vivo data (Fig. 1b), demonstrate that repetitive cerebral hyperperfusion secondary to transient hypertension leads to functional and structural changes in both the cerebrovascular endothelium (reduced dilatory responsiveness) and smooth muscle (increased reactivity/increased collagen deposition). We also demonstrated a 40% reduction in sympathetic innervation density of the cerebrovasculature when chronically exposed to repetitive autonomic dysreflexia (Fig. 3b).   38 2.4 Discussion  From both a functional and structural perspective, the cerebrovasculature is deleteriously impacted by chronic exposure to repetitive transient hypertension after SCI and the resulting cerebral hyperperfusion. The data showing that cerebral hyperperfusion occurs during transient hypertension in uninjured animals supports the contention that chronic exposure to transient hypertension may also be deleterious in those with SCI. The data showing that cerebral hyperperfusion occurs during transient hypertension in uninjured animals raises the concern that chronic exposure to transient hypertension may also be deleterious to the cerebrovasculature in uninjured models and populations, which is supported by epidemiological evidence, but not tested in the present study(Everson et al. 2001; Klonoff et al. 1989; Mostofsky et al. 2010). Similar transient elevations in blood pressure as those demonstrated in the present study occur in response to chronic caffeine consumption, high-intensity interval exercise, defecation, exaggerated blood pressure responses to mental stress, and drug abuse(Hansson et al. 1998; Lucas et al. 2015; Phillips et al. 2013; Resnick et al. 1977; Vlachopoulos et al. 2004).  The observed endothelial dysfunction and profibrotic stiffening plays a key role in the development of cerebrovascular complications and therefore likely underpins the association between the majority of the mentioned behaviors/conditions and cerebrovascular diseases. Repetitive transient hypertension is clearly a unique form of cerebrovascular decline compared with that resulting from chronic steady-state hypertension, as demonstrated by the lack of cerebrovascular inward remodeling (Fig. 2c, 2d) and no ensuing reduction in CBF (Fig. 3d), as well as the absence of elevated cerebrovascular myogenic tone(Gibbons and Dzau 1994).  The present data, demonstrating impaired endothelial function, inappropriate vasoconstrictive reactivity, as well as profibrotic stiffening of the cerebrovasculature, because of  39 transient hypertension, undoubtedly contributes to our previous observations of increased risk of stroke after SCI(Cragg et al. 2013). Further, the pathophysiological changes observed because of autonomic dysreflexia in this study provide a physiological rationale for the impairment of both neurovascular coupling (i.e., hyperemic response to neuronal activation) and autoregulation we have shown after SCI, because appropriate dilation, constriction, and distensibility of the cerebrovasculature are crucial for both of these cerebrovascular regulatory mechanisms(Phillips et al. 2013, 2015; Phillips, Krassioukov, et al. 2014; Phillips, Warburton, et al. 2014; Willie et al. 2014). In support of this, very recently we showed that autonomic dysfunction was associated with the development of vascular-cognitive impairment after SCI(Phillips et al. 2017).  Our previous work has also demonstrated that SCI results in an array of cerebrovascular consequences including profibrotic remodeling as well as loss of sympathetic regulation, which may make this population more susceptible to cerebrovascular trauma during transient hypertension because of impaired capacity to regulate CBF(A. A. Phillips et al. 2016; Phillips et al. 2017). Clearly, it would be beneficial in the clinical setting to prevent the cerebrovascular consequences of repetitive transient hypertension after SCI by reducing the burden of autonomic dysreflexia. We have previously demonstrated that some candidate prevention strategies may include early initiation of passive lowerlimb exercise after SCI or prophylactic antihypertensive administration with nifedipine or prazosin(Phillips and Krassioukov 2015). Chronic exposure to transient hypertension also led to profibrotic stiffening of the cerebrovasculature, which is well described in chronic steady-state hypertension but novel to the presently studied model(Baumbach and Heistad 1988; Gibbons and Dzau 1994). The vascular stiffening (and resistance to vasomotion) will also contribute to the observed impaired endothelial dilation, but also likely masks even more severe vasoconstrictive hypersensitivity. Intriguingly,  40 transient hypertension, as opposed to chronic hypertension, did not lead to inward remodeling of the vasculature, or the resulting reduction in CBF (Fig. 3C) because of increased vascular resistance, demonstrating that transient hypertension may represent a unique form of cerebrovascular decline not yet identified.  The large reduction in sympathetic innervation density of the cerebrovasculature from chronic exposure to transient hypertension (Fig. 3b), taken together with our previous work showing intact sympathetic pathways from pre-ganglionic neurons leading to the cerebrovasculature after T3 spinal cord transection, suggests that baroreceptor-mediated sympathetic withdrawal because of elevations in blood pressure is leading to reduced sympathetic control over the cerebrovasculature(A. A. Phillips et al. 2016). Because the sympathetic nervous system functions primarily to increase end-organ vascular resistance, reduced sympathetic control will contribute to the loss of capacity to autoregulate CBF after SCI, and paradoxically impair the brain’s ability to protect itself (through increased vascular resistance) from increases in brain perfusion pressure(Brassard, Tymko, and Ainslie 2017). These data provide insight into the mechanisms underlying our previous clinical research demonstrating that cerebral autoregulation is impaired in spinal cord injured patients with autonomic dysreflexia, and the increased risk of stroke in this population(Cragg et al. 2013; Phillips, Krassioukov, et al. 2014).  The CBF passively increased during transient hypertension in uninjured and injured animals, as well as humans with SCI, which supports the recently established contention that cerebral autoregulation is not as effective as thought previously(Willie et al. 2014). Spinal cord injured animals had slightly greater elevations in blood flow for a given increase in blood pressure, findings that suggest an impairment of cerebral autoregulation in the spinal cord injured animals  41 (Fig. 1a,1b,1c), and concur our previous clinical research(Phillips et al. 2017; Phillips, Krassioukov, et al. 2014).  This study provided important clinically relevant insight into the association between chronic exposure to transient hypertensive episodes because of autonomic dysreflexia, and cerebrovascular decline after SCI. Some perceived limitations may be that: (1) This study utilized transcranial Doppler to evaluate CBF, which measures velocity of red blood cells and assumes a constant diameter of the vessels being insonated. Other options, such as duplex extracranial ultrasound and MRI were not conducive to be used in a clinical setting such as that of the present study, because they require more personnel to operate as well as significant offline analysis and specialized equipment.33 (2) We did not assess females in the present study, because they are far less common to have a SCI.26 (3) We insonated the middle cerebral artery and not the posterior cerebral artery, so our findings do not extend to the posterior brain circulation, which may exhibit differential regulation as to the middle cerebral artery under some scenarios(Phillips et al. 2017; Phillips, Krassioukov, et al. 2014; Phillips, Warburton, et al. 2014). 2.5 Clinical perspectives  Stroke is 300–400% more likely to occur after SCI and is a leading cause of death. Autonomic dysreflexia, which leads to transient hypertension, affects the majority of persons with SCI up to 40 times per day. Acutely, we know that severe episodes of hypertension secondary to autonomic dysreflexia can lead to stroke; however, we do not understand the long-term consequences of this stimulus on the brain and its vasculature. The present translational study first demonstrated that our model of transient hypertension, secondary to autonomic dysreflexia, leads to substantial acute increases in CBF that were mimicking the responses in patients with SCI.   42 Next, we showed that chronic exposure to this stimulus leads to broad deleterious consequences to the brain vasculature, including endothelial dysfunction and profibrotic remodeling, and provided insight into the molecular mechanisms underpinning these changes (Fig. 3e). Although our findings were in a SCI model, these results may also provide insight into the association between stroke and a number of conditions where there is long-term exposure to transient hypertension, such as exaggerated mental stress, white coat syndrome, and stimulant drug abuse.  It appears that repeated transient elevations in blood pressure are not innocuous to the cerebrovasculature; this link has recently been highlighted in the popular media and questioned in the scientific literature(Lucas et al. 2015; Rothwell et al. 2010). It may be that exposure to repetitive transient hypertension over time is a critical risk factor for cerebrovascular diseases, and certainly future research into this is warranted, because it has also been suggested that blood pressure oscillations may exert neuroprotective effects during ischemic episodes(Ryan et al. 2011). It should be considered that the human brain has been shown to demonstrate a form of hysteresis, where cerebral autoregulation is more effective when faced with acute elevations in blood pressure compared with acute decreases(Brassard, Ferland-Dutil, et al. 2017; Tzeng et al. 2010). As such, it would be prudent to also explore the cardiovascular effects of chronic exposure to orthostatic hypotension, which is widespread after SCI and is epidemiologically linked to cardiovascular morbidity and death in nonspinal cord injured populations, and as such may be exerting even more severe cerebrovascular consequences as transient hypertension. Finally, this study provides evidence that autonomic dysreflexia needs to be aggressively managed after SCI(Dobkin 1989; Eigenbrodt et al. 2000; Rose et al. 2006).   43  Table 2-1. List of primary antibodies used for morphological assessment of middle cerebral arteries. Primary Antibody Antibody Concentration Reference Rabbit α Collagen I 1:500 Abcam, (ab34710); USA Mouse α Collagen III 1:300 Abcam, (ab6310); USA Rabbit α Elastin 1:200 Millipore, (AB2039); USA Rabbit α SMA 1:400 Abcam, (ab5694); USA Rabbit α TRPV4 1:300 Generous gift from Dr. Stefan Hellar at Stanford SMA, Smooth Muscle Actin; TRPV4, Transient receptor potential cation channel subfamily V member 4   44                   Figure 2-1. Developing a clinically-relevant preclinical model of transient cerebral hyper-perfusion secondary to autonomic dysreflexia.  A) Cerebral hyper-perfusion during 12 episodes of transient hypertension secondary to autonomic dysreflexia in six patients with chronic spinal cord injury above the 6th thoracic spinal segment. Clearly, there was little to no cerebral autoregulation during transient hypertension, as the increases in middle cerebral artery (MCA) cerebral blood flow was strongly linearly related to increased blood pressure. This certainly challenges the dogma that cerebral autoregulation is capable of maintaining cerebral blood flow constant in the face of acute bouts of hypertension.  B) Identical profile of cerebral hyper-perfusion during 12 episodes of transient hypertension during autonomic A  B  C  MCA  45 dysreflexia induced by colorectal distension in four Wistar rats with a T3 complete spinal cord transection. *Note the similar temporal and amplitudinal profile. C) Timeline of experiments. Colorectal distention began 14 days post spinal cord injury (SCI). Outcome measures took place after four weeks of colorectal distension and included pressure myography, magnetic resonance imaging, immunohistochemistry, and quantitative polymerase chain reaction.  46    Figure 2-2. Chronic exposure to transient cerebral hyper-perfusion impaired middle cerebral artery endothelial function, exacerbated stiffening, and led to hyper-sensitivity to constrictor stimuli after SCI.  Compared to T3-SCI (n=13), the exposure to repetitive colorectal distention (T3-SCI+CRD; n=15) led to reduced maximal dilation to carbachol (logECmax; A), but not sensitivity (logEC50; B). Compared to T3-SCI (n=13), the exposure to repetitive colorectal distention (T3-SCI+CRD; n=15) trended to result in altered mechanical properties of the MCA, such that distensibility (C) and strain (D) was reduced, whereas beta stiffness trended to be increased (H). Compared to T3-SCI (n=13), the exposure to repetitive colorectal distention (T3-SCI+CRD; n=15) did not lead to remodelling, such that lumen diameter, wall thickness and wall-to lumen ratio were similar between groups when using repeated measures ANOVA (E-G). Compared to T3-SCI (n=13), the exposure to repetitive colorectal distention (T3-SCI+CRD; n=15) led to hyperconstriction in  47 response to 10-5 M serotonin (I). CSA, cross-sectional area.   A,B,H,I) unpaired t-test.  C-G), repeated measures ANOVA (main effect), comparisons were made using physiological pressure 80-180 mmHg. A,B,H,I), analysis was performed at an intraluminal pressure of 80 mmHg. *Significantly different from T3-SCI (n=5; p<0.05). Values are mean ± SEM.    48  Figure 2-3. Endothelial mechanoreceptor overexpression, profibrosis, and loss of sympathetic perivascular innervation density in response to chronic exposure to repetitive transient hypertension.   A) The immunofluorescence intensity of Collagen I was elevated in the T3-SCI+CRD, indicating exacerbated profibrosis. Chronic exposure to repetitive transient hypertension also resulted in greater endothelial expression of the mechano-receptors transient receptor potential cation channel subfamily V member 4 (TRPV4). B) mRNA levels within the middle cerebral artery. C) Compared to T3-SCI, the exposure to repetitive colorectal distention led to reduced sympathetic innervation density, indicating sympathetic withdrawal from the cerebrovasculature. D) Representative immunohistochemical images from T3-SCI and T3-SCI+CRD animals for Collagen I, III, Elastin, smooth muscle actin (SMA), and tyrosine hydroxylase (TH). and TRPV4.  The top region of interest (ROI) was used for Collagen I, III, Elastin, SMA analysis of the media layer of the middle cerebral artery, while the bottom ROI was used to evaluate expression of  49 TRPV4 specific to the endothelium. E) There were no regional or global differences in cerebral blood flow as derived from magnetic resonance imaging. F) Schematic illustrating that cerebral hyper-perfusion secondary to transient hypertension leads to increased collagen expression in the vascular smooth muscle (red), endothelial dysfunction and overexpression of endothelial mechanoreceptors (purple), as well as hypersensitivity to constrictive stimuli and loss of sympathetic fibre density (blue). ***Significantly different from T3-SCI (p<0.001), **Significantly different from T3-SCI (p<0.01), *Significantly different from T3-SCI (p<0.05). Unpaired t-test. CBF, cerebral blood flow; TGF-β, transforming growth fa-beta; MMP-9, matrix metalloproteinase 9.    50 Chapter 3. Vascular-cognitive impairment following high-thoracic spinal cord injury is associated with structural and functional maladaptation in cerebrovasculature  3.1 Introduction Debilitating cardiovascular impairments are among the leading causes of morbidity and mortality in individuals with SCI (Moriarity et al., 2004; Garshick et al., 2005). Disruption of sympathetic cardiovascular control in individuals with upper thoracic or cervical SCI results in severe bidirectional fluctuations in systemic blood pressure (BP), which occur on a daily basis (Sachdeva et al., 2019). Chronic concomitant exposure to these extreme hypotensive and hypertensive events (called orthostatic hypotension and autonomic dysreflexia, respectively) following SCI can alter cerebrovascular structure and function, leading to cognitive decline. Not surprisingly, SCI is associated with 3-4 fold higher risk of stroke and a significantly greater risk of cognitive impairment (Cragg et al., 2013; Sachdeva et al., 2018). From our review of 38 studies, up to 60% of individuals with SCI exhibit impairments in one or more cognitive domains such as memory, attention, concentration or executive function (Sachdeva et al., 2018). Although cognitive impairment following SCI can be attributed to a number of comorbidities, considerable evidence substantiates the relationship between cerebrovascular dysfunction and cognitive impairment. Furthermore, the dysfunctions could be more pronounced following SCI as cardiovascular disease progression is accelerated in this population owing to the amplification of various physical, physiological, and environmental factors (Phillips & Krassioukov, 2015).  51 Multiple studies from our group and others have presented a relationship between cerebrovascular dysfunction and cognitive decline in individuals with SCI (Phillips et al., 2014a; Phillips et al., 2014b; Wecht et al., 2016). Intriguingly, cerebrovascular function is only partially restored in response to normalization of systemic BP, indicating that other factors (e.g. vascular remodeling, fibrosis, and endothelial dysfunction) may contribute to the observed vascular impairment associated with SCI. We have previously used a rat model to demonstrate that high-thoracic SCI leads to significant fibrosis and increased stiffness of cerebrovasculature at the sub-acute stage (Phillips et al., 2016). Understanding the progression of these early deleterious effects is of great clinical significance as this will potentially reveal novel therapeutic targets to prevent or mitigate chronic cerebrovascular and cognitive decline following SCI. The purpose of this study is to test if vascular-cognitive impairment occurs in a chronic experimental model of SCI with potential applications for the development of therapeutic interventions. Here, we used vascular physiological and immunohistochemical assessments to investigate the structural and functional correlates of cerebrovascular dysfunction after SCI. We also evaluated the impact of SCI on resting cerebral blood flow (CBF) and short-term memory in this model. We hypothesized that chronic high-thoracic SCI may produce cognitive deficits associated with diminished vascular reactivity, structural changes in the middle cerebral artery (MCA), and reduced CBF. 3.2 Methods 3.2.1 Experimental design All experimental procedures were carried out in accordance with the Guide for the Care and Use of experimental animals established by the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee (approval certificate number: A18-0183). Twenty-eight adult (250-300g) male Wistar rats were assigned into either a  52 control (CON, n=14) or T3-complete spinal cord transection group (SCI, n=14). Fourteen weeks post-injury, all rats were assessed for short-term memory using a novel object recognition test (NORT, Figure 1). Following the NORT, the groups were subdivided equally (n=7 each). Each subgroup was assigned for either in vitro physiological assessments using pressure myography or for magnetic resonance imaging (MRI) followed by immunohistochemistry.   3.2.3 T3 Transection surgery and animal care Surgery and animal care were conducted as described previously (Phillips et al., 2016). Rats were pre-treated with enrofloxacin [Baytril; 10 mg/kg, s.c., Associated Veterinary Purchasing (AVP), Langley, British Columbia, Canada] for three days. The animals were fasted the night before surgery. On the day of surgery, anesthesia was induced with 5% isoflurane (AErrane; AVP) in 100% oxygen at a flow rate of 1L/min and maintained at 2-3% during the surgery. Enrofloxacin (10 mg/kg, s.c.), buprenorphine (Temgesic; 0.02 mg/kg, s.c., University of McGill Animal Resources Centre), and lactated Ringer’s solution (5 mL, s.c.)  were administered preoperatively.  A dorsal midline incision was made through the skin and superficial muscle layer, as well as a blunt dissection of deep muscles overlying T1-T4 vertebrae. Deep muscles around T2 - T3 vertebrae were carefully dissected to expose the intervertebral gap. The dura at the gap was then opened, and the spinal cord was transected with microscissors. Complete transection was confirmed by two surgeons via visual separation of the rostral and caudal spinal cord stumps under the microscope.  Absorption sponges and Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, USA) were used to achieve hemostasis. The muscle and skin of the surgical site were closed with 4–0 Vicryl and 4–0 Prolene sutures, respectively. Animals received warmed lactated Ringer’s solution (5 mL, s.c.) and were allowed to recover from anesthesia in a temperature-controlled environment (Animal Intensive Care Unit, HotSpot for Birds, Los Angeles, CA, USA).  53 Enrofloxacin (10 mg/kg, s.c., once per day), buprenorphine (0.02 mg/kg, s.c., twice per day) were administered for 3 days post-surgery.  Animals with SCI were housed in specialized cages, with rubber matting to facilitate movement, low-reaching water bottles, and food scattered on the cage bottom to encourage foraging. Animals were supported with an enriched diet, including meal replacement shake (Ensure, Abbott Laboratories, Abbott Park, Illinois, USA), fruit, spinach, HydroGel (Clear H2O, Westbrook, ME, USA), peanuts and kibble (LabDiet, Rodent Diet 5001; PMI International, St. Louis, MO, USA). The bladder was manually emptied three times daily until spontaneous bladder function returned (~ 10 days after injury). Health status was monitored and recorded daily for the first 2 weeks after surgery and on alternative days thereafter. All experiments and analyses were performed with the investigators completely blinded of the group assignments.  3.2.4 NORT Short-term memory was evaluated using the NORT, a widely-implemented test to assess retention memory and interest in exploring novel objects (Antunes & Biala, 2012). One day prior to the testing session, animals were individually placed in an empty container for one hour to encourage habituation. One hour before the test, animals were exposed to two identical objects for ten minutes in the same container, following which one object was replaced with a novel object (Ennaceur & Delacour, 1988; Hammond et al., 2004; Taglialatela et al., 2009; Gaskin et al., 2010). During the test, animals were allowed to explore the open field freely for five minutes while being videotaped. Discrimination index (D-index) was calculated by dividing the difference between the exploration time devoted to novel (TN) and Object 1 (T1)  divided by the total exploration time [DI = (TN – T1)/(TN + TF)*100] (Ennaceur & Delacour, 1988).    54 3.2.5 CBF CBF maps were acquired using arterial spin labeling (ASL) magnetic resonance imaging (MRI) as previously described (Phillips et al., 2018). Briefly, water molecules from inflowing blood were magnetically excited by a spin inversion. The overall magnetization of a downstream area of the brain was modified upon the arrival of these labelled molecules. The resultant signal describes the amount of arterial blood delivered to the imaging slice within the transit time of the experiment. On the day of imaging, rats were sedated with isoflurane and maintained on a ventilator (Harvard Apparatus Inspira ASV, USA) to maintain expired CO2 at 35 mmHg. Body temperature was maintained at 37o C. Assessments were performed on a 7 Tesla MRI Scanner (Bruker Biospec 70/30 USR, Bruker, Ettlingen, Germany) using a quadrature volume transmit coil and 4-channel phased-array receivers. Multiple inversion recovery echo-planar imaging (EPI) scans were acquired to obtain calibration data (tissue T1, inversion efficiency, and proton density) for the ASL scan (recovery time = 10 sec, echo time = 11.47ms, 22 inversion times between 30-8000ms spaced geometrically, field of view = 40x40 mm, matrix = 64x64, slice thickness = 2mm, 9 slices, single shot).  Flow-sensitive alternating inversion recovery EPI with a pre-saturation preparation (Wegener et al., 2007) was used to acquire the ASL data at five inversion times to allow correction for variable transit times using same geometry as the calibration scans. CBF maps were calculated by fitting the full perfusion model, including non-zero arterial transit time and bolus outflow (Buxton et al., 1998). 3.2.6 Pressure myography MCA function was assessed using pressure myography (Living Systems, St. Albans, VT, USA) as described previously (Pires et al., 2011; Phillips et al., 2016; Fan et al., 2017). A 5 × 3 mm section of brain tissue containing the MCA was removed and placed in Ca2+ free physiological salt solution (PSS) at 4°C, with 1% bovine serum albumin. A branch-free segment of the MCA was  55 dissected and mounted on two glass micropipettes in the pressure myograph, and the diameter was measured using a digital CCD camera. MCAs were equilibrated at 80 mmHg for 45 min in PSS containing (in mmol) 141.9 NaCl, 4.7 KCl, 1.12 KH2PO4, 1.7 MgSO4.7H2O, 2.8 CaCl2, 10 Hepes, 5 dextrose and 0.5 EDTA (pH=7.4) solution. The baseline of the inner diameter of the MCA was measured and only vessels that developed >20% spontaneous myogenic tone were used for the experiment. Passive distensibility was assessed in Ca2+‐free PSS containing 0.002 M EGTA and intraluminal pressure was increased from 20 to 180 mmHg in 20 mmHg increments. Endothelial function was assessed by measuring the response to administration of carbachol in the bath as previously described (Brueggemann et al., 2009; Wang et al., 2012). The MCA was first bathed in PSS and pressurized at 80 mmHg to generate a stable degree of spontaneous myogenic tone. The vasodilator response to increasing concentrations of carbachol (10-10 to 10-5M) was then determined (Dauphin & Hamel, 1990). To investigate the mechanism involved, the vessel was pre-incubated with a TRPV 4 antagonist, HC067047 (10-6 M), for 15 min and the responses to carbachol  (10-10 to 10-5 M) were recorded (Ho et al., 2015). Finally, the vessels were bathed in Ca2+-free PSS and a passive pressure diameter curve was recorded. Myogenic tone was calculated using the following formula: % tone = [1 − (active lumen diameter/ passive lumen diameter)] × 100.  3.2.7 Perfusion and tissue collection Animals were euthanized with isoflurane followed by intraperitoneal injection of chloral hydrate. The cerebral vasculature was fixed by transcardial perfusion with 100-150 ml of phosphate-buffered saline (at 80-90 mmHg) and 400 ml of 4% paraformaldehyde at 120-140 mmHg. The brain was harvested and placed in 4% paraformaldehyde for 24 hours at 4°C, and then transferred to 20% sucrose for three days. A 2-3mm long segment of MCA was gently dissected from each hemisphere and used for histological analyses.   56 3.2.8 Immunohistochemistry  A segment of the perfused MCA from each animal was embedded in Cryomatrix® and 10 µm frozen transverse sections were cut using a cryostat. The sections were placed on Superfrost Plus slides and stored at -80ºC until processed for immunohistochemistry.  Briefly, MCA sections were washed in phosphate buffer saline (PBS) and 0.1 M PBS containing 0.3% Triton X-100 (PBST). They were blocked for 1 h in 10% normal donkey serum in PBST. Sections were then incubated with the primary antibodies to: collagen I (COL I, 1:500, Abcam, ab34710), collagen 3 (COL III, 1:300, Abcam, ab6310), TRPV4 (1:300, generous gift from Dr. Stefan Heller lab at Stanford), CD31 (10µg/mL, R&D systems, AF3628), and a smooth muscle actin (SMA, 1:400, Abcam, ab5694) overnight at room temperature. On the second day, sections were washed in PBST and incubated with species-specific secondary antibodies: Alexa™-fluor 488 donkey anti-mouse (1:1000; Molecular Probes©, USA), Alexa™-fluor 488 donkey anti-goat (1:1000; Molecular Probes©, USA) or Cy3 conjugated donkey anti-rabbit IgG antibody (1:1000; Jackson ImmunoResearch) for 1h. Finally, sections were washed with PBST, coverslipped with Vectashield mounting medium (Vector Laboratories Inc.) and images were captured using a confocal microscope with identical acquisition parameters. Quantification  The intensity of immunoreactivity of MCAs were assessed using the Fiji Software (http://fiji.sc/Downloads#Fiji). Staining intensities were averaged from 4 sections per animal. For the analyses of COL I, COL III and SMA, the region of interest (ROI) was confined between the adventitial border of the artery and the lumenal interface. TRPV 4 and CD31, were specifically quantified in the endothelial layer (Figure 4). Furthermore, a negative control consisting of staining with secondary antibody in the absence of primary antibody was performed for each antibody (Frias et al., 2015; Phillips et al., 2016).   57 3.2.9 Morphometric assessment of the MCA The arterial wall thickness and lumen diameter were measured using the transverse sections of MCA immunostained with SMA (four sections per rat). Within each section, four sites were selected each representing a quadrant of the vessel, and a line was drawn across the media of the vascular wall perpendicular to the tangent line, and the length of this line was measured. The average of the four measurements was used to denote wall thickness for each section. Lumen diameter was measured by taking the average of the widest and the narrowest part of the lumen. 3.2.10 Tyrosine hydroxylase (TH) staining of the MCA whole mounts A small segment (3-4 µm) from the middle third of MCA was stained for tyrosine hydroxylase (1:1000; anti-rabbit; Millipore; AB152) to compare the degree of sympathetic innervation. The density of TH-positive (TH+) nerve fibers was measured using Fiji Software (based on ImageJ, http://fiji.sc/Downloads#Fiji). A grid was generated over the entire image (44 um2/grid sector). A 5x5 ROI was randomly selected, and the intersections between the grid and the stained fibers were counted. The number of crossings of fibers with the grid was used to determine the density of TH+ fibers that innervate the middle third of MCA as previously described (Hesp et al., 2012; Phillips et al., 2015b).  3.2.11 Statistical analyses Mean values ± standard error are presented. The significance of differences between mean values in the control and spinal cord transected groups were determined using a Student's t-test or a nonparametric alternative when the data was not normally distributed. Repeated measures ANOVA was used for concentration–response curves for carbachol, which were further analyzed by nonlinear regression (Hill equation) to calculate EC50 and Emax values. All the statistical analyses were carried out using GraphPad Prism 7.0 software (GraphPad, San Diego, CA). A p value of less than 0.05 was considered to be statistically significant.  58 3.3 Results 3.3.1 Altered endothelial function and mechanical properties of the MCA after chronic SCI The endothelial function of the MCA was impaired in the rats with SCI (Figure 2). The maximal vasodilation response to carbachol was 55% less in the SCI group than in the uninjured controls (Figure 2A, B), but the EC50 was not significantly different (Figure 2C, D). Furthermore, when tested across a range of intralumenal pressures in Ca2+ free solution, the distensibility of MCA was significantly reduced, with 37% reduction at 80 mmHg (Figure 2E). The difference in endothelium-mediated vasodilation response between the two groups was likely mediated via endothelial TRPV 4, as the response to carbachol was similar in both groups following treatment with the TRPV 4 channel antagonist HC067047 (Figure 2A & B). This was further supported by immunohistochemical analysis, which demonstrated that endothelial TRPV 4 expression was 46% lower in rats with SCI (Figure 2G). Furthermore, a 34% lower expression of endothelial CD31 in the SCI is suggestive of loss of endothelial cells or changes in their function (Figure 2G).  3.3.2 Morphological and structural changes in the MCA after SCI Immunohistochemical analyses revealed there is a marked 50% increase in COL I deposition in the media of the MCA after SCI (Figure 3A). This is associated with a significant reduction in SMA staining. However, there was no significant difference in wall thickness, lumen diameter, or wall to lumen ratio (Figure 3B). Perivascular sympathetic innervation of MCAs, as revealed by TH+ axonal density, was also not affected by SCI (Figure 3C). 3.3.3 Chronic SCI is associated with a reduction in CBF Resting CBF was 32% lower in T3-SCI (Figure 4A, B), indicating reduced brain perfusion following chronic SCI. Moreover, regional flow in SCI was significantly reduced in the  59 hippocampus (32%, Figure 4C). No regional effects were seen on CBF in the basal forebrain or cortex. 3.3.4 SCI disrupts short-term memory Short-term memory was analyzed using the NORT, a method that is relatively less dependent on locomotor activity (Figure 4D) (Wu et al., 2014). The control rats showed a higher preference for the novel object (D index= 81.1 ± 13.9). In contrast, the rats with chronic SCI had significantly reduced preference to the novel object (D index= 62.6 ± 19.2), suggesting impairment in short-term retention memory.  3.4 Discussion This study provides a novel mechanistic evidence of vascular cognitive impairment in a model of SCI that consistently results in highly labile systemic BP. Although a number of clinical studies in the recent years have demonstrated associations between disrupted cardiovascular control and cognitive impairment following SCI, the majority of them are unable to rule out the contribution of a concomitant traumatic brain injury [reviewed in (Sachdeva et al., 2018)]. The present results indicate that chronic SCI results in structural and functional impairments in the MCA, namely: endothelial dysfunction, elevated expression of COL I (the major collagen isoform in the media of vascular wall) and significantly impaired MCA distensibility. Consistent with the clinical literature (Catz et al., 2008; Wecht et al., 2016), we show that experimental high-thoracic SCI results in significant cerebral hypoperfusion. Our comprehensive review has concluded that regulation of CBF following SCI is drastically impacted via a number of factors including endothelial dysfunction (Phillips et al., 2013). Interestingly, the reduction in CBF in the present study was observable in the hippocampus, a region associated with memory and likely more susceptible to ischemic damage due to its circulatory pattern (Roman, 2004). As hypothesized, a high-thoracic SCI that is known to cause detrimental cardiovascular and cerebrovascular impairments, resulted  60 in significant impairments in memory. Retention memory was assessed using the NORT, which is relatively less dependent on locomotion compared to other pre-clinical tests of cognitive function and has been previously employed in rats with SCI (Wu et al., 2014). Endothelial dysfunction was identified as a prominent outcome in chronic SCI, as indicated by the impaired response to carbachol. This is an important observation in light of our previous findings that endothelial function remains unaltered in the sub-acute stage of SCI (Phillips et al., 2016). This finding may indicate a therapeutic time window for early intervention that may be capable of preventing or impeding the ensuing decline in endothelial function, CBF and cognitive ability - an evident characteristic of chronic SCI. Endothelium-dependent relaxation, as induced by carbachol is primarily mediated via three pathways: nitic oxide (NO), prostacyclin and/or endothelial-derived hyperpolarization factor (EDHF) (Ozkor & Quyyumi, 2011). Particularly in the MCA, carbachol activates the M3 muscarinic receptor in endothelial cells that opens TRPV 4 channels, resulting in calcium influx which increases the formation of  NO and EDHF (Filosa et al., 2013).  Therefore, blockade of TRPV 4 channels suppresses NO and EDHF-mediated vasodilation (Marrelli et al., 2007; Zhang & Gutterman, 2011; Sukumaran et al., 2013). Interestingly, the administration of a TRPV 4 channel inhibitor had a greater effect to reduce the vasodilator response to carbachol in control animals than SCI animals, while the residual response to carbachol remained similar in both groups. This is consistent with endothelial dysfunction and reduced production of NO in the SCI rats. Therefore, the underlying mechanism behind SCI-derived loss of endothelial reactivity may be related to a reduction in TRPV 4 channel activity. This argument is futher supported by our immunohistochemical data, where the expression of endothelial TRPV 4 is reduced following SCI. Because TRPV 4 channels are sensitive to shear stress, the abnormal expression of endothelial TRPV 4 receptors after T3-SCI may be associated  61 with the global reduction in CBF (Filosa et al., 2013). Furthermore, decreased expression of CD31 suggests potential structural damage to the MCA endothelium following SCI, consistent with other reports of reduced CD31 in models of cognitive impairment associated with ischemia (Plaschke et al., 2008). In addition to endothelial dysfucntion, SCI resulted in a marked increase in COL I deposition in the MCA and reduced distensibility, suggesting stiffer arteries. While previous literature implies a role of disrupted sympathetic innervation in increasing fibrosis in the aorta (Fronek et al., 1978), it is unlikely that a similar mechanism is responsible for the stiffening of the MCA in the present study. This is supported by the near-identical TH staining in the MCAs of the two experimental groups. Moreover, considering the chronic increased BP variability following disruption of sympathetic control, the role of neuro-humoral processes in mediating arterial stiffening via increased pro-inflammatory factors should be investigated as a possible underlying mechanism (Wang et al., 2016). 3.5 Future perspectives We present an experimental high-thoracic SCI model that leads to endothelial dysfunction, structural remodeling of the cerebral arteries, global reduction in CBF and vascular-cognitive impairment, which has now been identified as a serious disease burden in individuals with SCI (Jegede et al., 2010; Phillips et al., 2014b; Phillips & Krassioukov, 2015; Phillips et al., 2018). Interestingly, there appears to be a therapeutic time window between the establishment of vascular fibrosis and onset of cognitive symptoms, offering various directions for upcoming research. Future studies could explore a number of intriguing avenues, such as spatiotemporal changes in expression of specific receptor subtypes to facilitate testing of potential drugs and other interventions for early prevention of vascular-cognitive impairment following SCI. For instance,  62 upregulation of TRPV4 channel activity may be a viable therapy for mitigating loss of cerebrovascular function.  JNc-440, a designed molecule to strengthen the interaction between TRPV 4 and activated potassium channel 3 (KCa2.3), has been recently reported to enhance endothelial-induced vasodilation and exert anti-hypertensive effects (He et al., 2017). A possible clinically-relevant intervention could also be physical exercise, which has been shown to have significant benefits on endothelial function (Fuchsjager-Mayrl et al., 2002; Di Francescomarino et al., 2009). Moreover, a better understanding of SCI-induced structural and functional changes in cerebral microvessels is needed. Alterations in endothelial health of microvessels will likely provide deeper insight into the underlying mechanisms of vascular-cognitive impairment after chronic SCI and provide novel therapeutic targets (Yu et al., 2015).    63   Figure 3-1. Experimental timeline.  The rats were divided into two experimental groups (n=14 each); CON (uninjured) and SCI (complete transection at T3 spinal segment). At fourteen weeks post-SCI (and age-matched controls), all rats underwent NORT. Following NORT, the groups were subdivided equally and assigned to (1) in vitro physiological assessments using pressure myography and, (2) MRI followed by immunohistochemistry.  64   Figure 3-2. Carbachol-induced relaxation is diminished in MCA after chronic SCI, which is closely associated with the loss of TRPV 4 and CD31 staining in the endothelium.  A & B. Dose-response curve of MCA in control (CON n=6) and rats with SCI (SCI, n=6) to carbachol. C. Maximal dilation of MCAs to carbachol. D. logEC50 to carbachol. E. MCA distensibility curve under Ca2+ free solution. F. Representative images of transverse sections of the MCA immunostained for CD31 and TRPV 4 illustrating co-localization of the fluorescent signals in the endothelium (arrows). G. Comparison of the immunofluorescence staining of  CD31 and TRPV 4 in endothelial layer of MCAs in control and SCI rats. Values are  65 mean±standard error; A&B., **** significantly different from CON using a one-way ANOVA with Tukey’s post-hoc test (p< 0.0001); E & G, *significantly different from CON (p < 0.05) using an unpaired t-test  66      Figure 3-3. Chronic SCI leads to increased collagen deposition in the arterial wall.  A. immunofluorescence intensity of proteins associated with fibrosis between CON and SCI. Values are mean ± SEM. Intensity for COL I was increased by 50%. No change was seen in Col III and SMA. * indicates significantly different from CON (p< 0.05); unpaired t-test. B. No morphological change was found in MCA passive structure. C. TH+ axon density does not change after chronic SCI. Arrows indicate TH-positive axons.  67   Figure 3-4. Reduced baseline CBF and impaired spatial short-term memory are observed after prolonged high-thoracic SCI.  A. Representative MRI images and CBF heat maps from the controls (CON) and rats with SCI (SCI). Major brain regions associated with memory are labelled in T2 scan images (cortex in red,  68 hippocampus in green, and basal forebrain in purple.) B. Global CBF is reduced after chronic SCI, indicating lack of blood perfusion to the brain. C. Brain regions associated with memory show a trend of reduced CBF after chronic SCI. D. D-index of the NORT is significantly reduced in the SCI group. Injured animals exhibit significantly less of a preference for the novel object compared to controls. * indicates significantly different from CON (p < 0.05); unpaired t-test. Values are mean±SEM. CON, uninjured animals, SCI, T3-transected animals, CBF, cerebral blood flow. ASL, arterial spin labeling.    69 References Alan N, Ramer LM, Inskip JA, Golbidi S, Ramer MS, Laher I & Krassioukov A V. (2010). Recurrent autonomic dysreflexia exacerbates vascular dysfunction after spinal cord injury. Spine J 10, 1108–1117. Alessandri N, Tufano F, Petrassi M, Alessandri C, Lanzi L, Fusco L, Moscariello F, De Angelis C & Tomao E (2010). Elasticity/distensibility of the ascending aorta: basal conditions and simulated conditions from space flights. Eur Rev Med Pharmacol Sci 14, 421–426. Antunes M & Biala G. (2012). The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13, 93-110.  Baumbach, Gary L. and Donald D. Heistad. 1988. “Cerebral Circulation in Chronic Arterial Hypertension.” Hypertension 12(2):89–95.  Baumbach GL & Hajdu MA (1993). Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats. Hypertension 21, 816–826. Biering-Sørensen, F., M. Craggs, M. Kennelly, E. Schick, and J. J. Wyndaele. 2008. “International Lower Urinary Tract Function Basic Spinal Cord Injury Data Set.” Spinal Cord 46(5):325–30.  Brassard, Patrice, Michael M. Tymko, and Philip N. Ainslie. 2017. “Sympathetic Control of the Brain Circulation: Appreciating the Complexities to Better Understand the Controversy.” Autonomic Neuroscience : Basic & Clinical.  Brock JA, Yeoh M & McLachlan EM (2006). Enhanced neurally evoked responses and inhibition of norepinephrine reuptake in rat mesenteric arteries after spinal transection. Am J Physiol Heart Circ Physiol 290, H398–H405. Brueggemann LI, Mackie AR, Mani BK, Cribbs LL & Byron KL (2009). Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles. Mol Pharmacol 76, 1053–1061. Buxton RB, Frank LR, Wong EC, Siewert B, Warach S & Edelman RR. (1998). A general kinetic model for quantitative perfusion imaging with arterial spin labeling. Magn Reson Med 40, 383-396. Chan S-L, Chapman AC, Sweet JG, Gokina NI & Cipolla MJ (2010). Effect of PPARγ inhibition during pregnancy on posterior cerebral artery function and structure. Front Physiol 1, 130. Charkoudian N, Joyner MJ, Sokolnicki LA, Johnson CP, Eisenach JH, Dietz NM, Curry TB & Wallin BG (2006). Vascular adrenergic responsiveness is inversely related to tonic activity of sympathetic vasoconstrictor nerves in humans. J Physiol 572, 821–827.  70 Claydon VE & Krassioukov A V (2008). Clinical correlates of frequency analyses of cardiovascular control after spinal cord injury. Am J Physiol Hear Circ Physiol 294, H668–H678. Coyle P (1987). Dorsal cerebral collaterals of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY). Anat Rec 218, 40–44. Catz A, Bluvshtein V, Pinhas I, Akselrod S, Gelernter I, Nissel T, Vered Y, Bornstein N & Korczyn AD. (2008). Cold pressor test in tetraplegia and paraplegia suggests an independent role of the thoracic spinal cord in the hemodynamic responses to cold. Spinal cord 46, 33-38.  Cragg JJ, Noonan VK, Krassioukov A & Borisoff J (2013). Cardiovascular disease and spinal cord injury: Results from a national population health survey. Neurology 81, 723–728. Dauphin F & Hamel E (1990). Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology. Eur J Pharmacol 178, 203–213. Davidoff GN, Roth EJ, Haughton JS & Ardner MS (1990). Cognitive dysfunction in spinal cord injury patients: sensitivity of the Functional Independence Measure subscales vs neuropsychologic assessment. Arch Phys Med Rehabil 71, 326–329. DeVivo MJ, Go BK & Jackson AB (2002). Overview of the national spinal cord injury statistical center database. J Spinal Cord Med 25, 335–338. Di Francescomarino S, Sciartilli A, Di Valerio V, Di Baldassarre A & Gallina S. (2009). The effect of physical exercise on endothelial function. Sports Med 39, 797-812.  Dobkin, B. H. 1989. “Orthostatic Hypotension as a Risk Factor for Symptomatic Occlusive Cerebrovascular Disease.” Neurology 39(1):30. Dorrance AM, Matin N & Pires PW (2014). The effects of obesity on the cerebral vasculature. Curr Vasc Pharmacol 12, 462–472. Duprez DA (2006). Role of the renin-angiotensin-aldosterone system in vascular remodeling and inflammation: a clinical review. J Hypertens 24, 983–991. Eigenbrodt, M. L., K. M. Rose, D. J. Couper, D. K. Arnett, R. Smith, and D. Jones. 2000. “Orthostatic Hypotension as a Risk Factor for Stroke: The Atherosclerosis Risk in Communities (ARIC) Study, 1987-1996.” Stroke 31(10):2307–13.  Ennaceur A & Delacour J. (1988). A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behavioural brain research 31, 47-59.  Everson, S. A., J. W. Lynch, G. A. Kaplan, T. A. Lakka, J. Sivenius, and J. T. Salonen. 2001. “Stress-Induced Blood Pressure Reactivity and Incident Stroke in Middle-Aged Men.” Stroke; a Journal of Cerebral Circulation 32(6):1263–70.   71 Fan F, Pabbidi MR, Ge Y, Li L, Wang S, Mims PN & Roman RJ. (2017). Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries. Am J Physiol Renal Physiol 312, F971-F981.  Filosa JA, Yao X & Rath G. (2013). TRPV4 and the regulation of vascular tone. J Cardiovasc Pharmacol 61, 113-119.  Fougere, Renée J., Katharine D. Currie, Mark K. Nigro, Lynn Stothers, Daniel Rapoport, and Andrei V Krassioukov. 2016. “Reduction in Bladder-Related Autonomic Dysreflexia after OnabotulinumtoxinA Treatment in Spinal Cord Injury.” Journal of Neurotrauma 33(18):1651–57.  Frias B, Santos J, Morgado M, Sousa MM, Gray SM, McCloskey KD, Allen S, Cruz F & Cruz CD. (2015). The role of brain-derived neurotrophic factor (BDNF) in the development of neurogenic detrusor overactivity (NDO). The Journal of Neuroscience : the official journal of the Society for Neuroscience 35, 2146-2160.  Fronek K, Bloor CM, Amiel D & Chvapil M. (1978). Effect of long-term sympathectomy on the arterial wall in rabbits and rats. Exp Mol Pathol 28, 279-289.  Fuchsjager-Mayrl G, Pleiner J, Wiesinger GF, Sieder AE, Quittan M, Nuhr MJ, Francesconi C, Seit HP, Francesconi M, Schmetterer L & Wolzt M. (2002). Exercise training improves vascular endothelial function in patients with type 1 diabetes. Diabetes Care 25, 1795-1801.  Furlan, Julio C., Michael G. Fehlings, William Halliday, and Andrei V Krassioukov. 2003. “Autonomic Dysreflexia Associated with Intramedullary Astrocytoma of the Spinal Cord.” The Lancet. Oncology 4(9):574–75.  Fronek K, Bloor CM, Amiel D & Chvapil M (1978). Effect of long-term sympathectomy on the arterial wall in rabbits and rats. Exp Mol Pathol 28, 279–289. Garshick E, Kelley A, Cohen SA, Garrison A, Tun CG, Gagnon D & Brown R (2005). A prospective assessment of mortality in chronic spinal cord injury. Spinal Cord 43, 408–416. Geary GG, Krause DN, Purdy RE & Duckles SP (1998). Simulated microgravity increases myogenic tone in rat cerebral arteries. J Appl Physiol 85, 1615–1621. Gibbons, G. H. and V. J. Dzau. 1994. “The Emerging Concept of Vascular Remodeling.” The New England Journal of Medicine 330(20):1431–38.  Grände G, Nilsson E & Edvinsson L (2013). Comparison of responses to vasoactive drugs in human and rat cerebral arteries using myography and pressurized cerebral artery method. Cephalalgia 33, 152–159. De Groot PC, Bleeker MW, van Kuppevelt DH, van der Woude LH & Hopman MT (2006). Rapid and Extensive Arterial Adaptations After Spinal Cord Injury. Arch Phys Med Rehabil 87, 688–696.  72 De Groot PCE, Poelkens F, Kooijman M & Hopman MTE (2004). Preserved flow-mediated dilation in the inactive legs of spinal cord-injured individuals. Am J Physiol Heart Circ Physiol 287, H374–H380. Groothuis J & Thijssen D (2010). Angiotensin II contributes to the increased baseline leg vascular resistance in spinal cord-injured individuals. J  …. Hammond RS, Tull LE & Stackman RW. (2004). On the delay-dependent involvement of the hippocampus in object recognition memory. Neurobiol Learn Mem 82, 26-34.  Handrakis JP, DeMeersman RE, Rosado-Rivera D, LaFountaine MF, Spungen AM, Bauman WA & Wecht JM (2009). Effect of hypotensive challenge on systemic hemodynamics and cerebral blood flow in persons with tetraplegia. Clin Auton Res 19, 39–45. Hansson, L., A. Zanchetti, S. G. Carruthers, B. Dahlöf, D. Elmfeldt, S. Julius, J. Ménard, K. H. Rahn, H. Wedel, and S. Westerling. 1998. “Effects of Intensive Blood-Pressure Lowering and Low-Dose Aspirin in Patients with Hypertension: Principal Results of the Hypertension Optimal Treatment (HOT) Randomised Trial. HOT Study Group.” Lancet (London, England) 351(9118):1755–62.  Hesp ZC, Zhu Z, Morris TA, Walker RG & Isaacson LG (2012). Sympathetic reinnervation of peripheral targets following bilateral axotomy of the adult superior cervical ganglion. Brain Res 1473, 44–54. He D, Pan Q, Chen Z, Sun C, Zhang P, Mao A, Zhu Y, Li H, Lu C, Xie M, Zhou Y, Shen D, Tang C, Yang Z, Jin J, Yao X, Nilius B & Ma X. (2017). Treatment of hypertension by increasing impaired endothelial TRPV4-KCa2.3 interaction. EMBO Mol Med 9, 1491-1503.  Ho WS, Zheng X & Zhang DX. (2015). Role of endothelial TRPV4 channels in vascular actions of the endocannabinoid, 2-arachidonoylglycerol. British journal of pharmacology 172, 5251-5264.  Hubli M, Gee CM & Krassioukov A V (2014). Refined Assessment of Blood Pressure Instability After Spinal Cord Injury. Am J Hypertens10.1093/ajh/hpu122. Izzard AS, Horton S, Heerkens EH, Shaw L & Heagerty AM (2006). Middle cerebral artery structure and distensibility during developing and established phases of hypertension in the spontaneously hypertensive rat. J Hypertens 24, 875–880. Jegede AB, Rosado-Rivera D, Bauman WA, Cardozo CP, Sano M, Moyer JM, Brooks M & Wecht JM (2010). Cognitive performance in hypotensive persons with spinal cord injury. Clin Auton Res 20, 3–9. Klonoff, D. C., B. T. Andrews, and W. G. Obana. 1989. “Stroke Associated with Cocaine Use.” Archives of Neurology 46(9):989–93.   73 Krassioukov A & Claydon VE (2006). The clinical problems in cardiovascular control following spinal cord injury: an overview. Prog Brain Res 152, 223–229. Leask A & Abraham DJ (2004). TGF-beta signaling and the fibrotic response. FASEB J 18, 816–827. Lucas, Samuel J. E., James D. Cotter, Patrice Brassard, and Damian M. Bailey. 2015. “High-Intensity Interval Exercise and Cerebrovascular Health: Curiosity, Cause, and Consequence.” Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism 35(6):902–11.  Lujan HL, Janbaih H & DiCarlo SE (2012). Dynamic interaction between the heart and its sympathetic innervation following T5 spinal cord transection. J Appl Physiol 113, 1332–1341. Lujan HL, Palani G & DiCarlo SE (2010). Structural neuroplasticity following T5 spinal cord transection: increased cardiac sympathetic innervation density and SPN arborization. Am J Physiol Regul Integr Comp Physiol 299, R985–R995. MacDougall, J. D., D. Tuxen, D. G. Sale, J. R. Moroz, and J. R. Sutton. 1985. “Arterial Blood Pressure Response to Heavy Resistance Exercise.” Journal of Applied Physiology (Bethesda, Md. : 1985) 58(3):785–90.  Marchesi C, Paradis P & Schiffrin EL (2008). Role of the renin-angiotensin system in vascular inflammation. Trends Pharmacol Sci 29, 367–374. Marrelli SP, O'Neil R G, Brown RC & Bryan RM, Jr. (2007). PLA2 and TRPV4 channels regulate endothelial calcium in cerebral arteries. American journal of physiology Heart and circulatory physiology 292, H1390-1397.  Montezano AC, Nguyen Dinh Cat A, Rios FJ & Touyz RM (2014). Angiotensin II and vascular injury. Curr Hypertens Rep 16, 431. Mostofsky, E., G. Schlaug, K. J. Mukamal, W. D. Rosamond, and M. A. Mittleman. 2010. “Coffee and Acute Ischemic Stroke Onset: The Stroke Onset Study.” Neurology 75(18):1583–88.  Noreau L, Shephard RJ, Simard C, Pare G & Pomerleau P (1993). Relationship of impairment and functional ability to habitual activity and fitness following spinal cord injury. Int J Rehabil Res 16, 265–275. O’Donnell, Martin J., Denis Xavier, Lisheng Liu, Hongye Zhang, Siu Lim Chin, Purnima Rao-Melacini, Sumathy Rangarajan, Shofiqul Islam, Prem Pais, Matthew J. McQueen, Charles Mondo, Albertino Damasceno, Patricio Lopez-Jaramillo, Graeme J. Hankey, Antonio L. Dans, Khalid Yusoff, Thomas Truelsen, Hans-Christoph Diener, Ralph L. Sacco, Danuta Ryglewicz, Anna Czlonkowska, Christian Weimar, Xingyu Wang, and Salim Yusuf. 2010. “Risk Factors for Ischaemic and Intracerebral Haemorrhagic Stroke in 22 Countries (the INTERSTROKE Study): A Case-Control Study.” Lancet 376(9735):112–23.  74  Ozkor MA & Quyyumi AA. (2011). Endothelium-derived hyperpolarizing factor and vascular function. Cardiol Res Pract 2011, 156146. Phillips AA, Ainslie PN, Krassioukov A V & Warburton DER (2013a). Regulation of Cerebral Blood Flow after Spinal Cord Injury. J Neurotrauma 30, 1551–1563. Phillips AA, Ainslie PN, Krassioukov A V. & Warburton DER (2013b). Regulation of Cerebral Blood Flow after Spinal Cord Injury. Phillips AA & Krassioukov AV. (2015). Contemporary Cardiovascular Concerns after Spinal Cord Injury: Mechanisms, Maladaptations, and Management. Journal of neurotrauma 32, 1927-1942.  Phillips AA, Krassioukov A V, Ainslie PN & Warburton DER (2014a). Perturbed and spontaneous regional cerebral blood flow responses to changes in blood pressure after high level spinal cord injury: the effect of midodrine. J Appl Physiol 116, 645–653. Phillips AA, Krassioukov A V, Zheng MMZ & Warburton DER (2013c). Neurovascular Coupling of the Posterior Cerebral Artery in Spinal Cord Injury: A Pilot Study. Brain Sci 3, 781–789. Phillips AA, Warburton DE, Ainslie PN & Krassioukov A V (2014b). Regional neurovascular coupling and cognitive performance in those with low blood pressure secondary to high-level spinal cord injury: improved by alpha-1 agonist midodrine hydrochloride. J Cereb Blood Flow Metab 34, 794–801. Phillips, A. A., P. N. Ainslie, A. V Krassioukov, and D. E. R. Warburton. 2013. “Regulation of Cerebral Blood Flow after Spinal Cord Injury.” Journal of Neurotrauma 30(18):1551–63.  Phillips, AA, Franco Hn Chan, Mei Mu Zi Zheng, Andrei V Krassioukov, and Philip N. Ainslie. 2015. “Neurovascular Coupling in Humans: Physiology, Methodological Advances and Clinical Implications.” Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism 36(4):647–64.  Phillips, Aaron A., Philip N. Ainslie, Darren E. R. R. Warburton, and Andrei V. Krassioukov. 2016. “Cerebral Blood Flow Responses to Autonomic Dysreflexia in Those with High Level Spinal Cord Injury.” Journal of Neurotrauma 33(3):315–18.  Phillips, Aaron A., Stacy L. Elliott, Mei MZ Zheng, and Andrei V Krassioukov. 2014. “Selective Alpha Adrenergic Antagonist Reduces Severity of Transient Hypertension during Sexual Stimulation after Spinal Cord Injury.” Journal of Neurotrauma 32(6):392–96.  Phillips AA, Matin N, Frias B, Zheng MM, Jia M, West C, Dorrance AM, Laher I & Krassioukov AV. (2016). Rigid and remodelled: cerebrovascular structure and function after experimental high-thoracic spinal cord transection. Journal of Physiology 594, 1677-1688.   75 Phillips AA, Matin N, Jia M, Squair JW, Monga A, Zheng MMZ, Sachdeva R, Yung A, Hocaloski S, Elliott S, Kozlowski P, Dorrance AM, Laher I, Ainslie PN & Krassioukov AV. (2018). Transient Hypertension after Spinal Cord Injury Leads to Cerebrovascular Endothelial Dysfunction and Fibrosis. Journal of neurotrauma 35, 573-581.  Pires PW, Dams Ramos CM, Matin N & Dorrance AM (2013). The effects of hypertension on the cerebral circulation. Am J Physiol Heart Circ Physiol 304, H1598–H1614. Pires PW, Rogers CT, McClain JL, Garver HS, Fink GD & Dorrance AM (2011). Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 301, H87–H97. Plaschke K, Staub J, Ernst E & Marti HH. (2008). VEGF overexpression improves mice cognitive abilities after unilateral common carotid artery occlusion. Exp Neurol 214, 285-292.  Pons M, Cousins SW, Alcazar O, Striker GE & Marin-Castaño ME (2011). Angiotensin II-induced MMP-2 activity and MMP-14 and basigin protein expression are mediated via the angiotensin II receptor type 1-mitogen-activated protein kinase 1 pathway in retinal pigment epithelium: implications for age-related macular degeneration. Am J Pathol 178, 2665–2681. Ramsey JBG, Ramer LM, Inskip JA, Alan N, Ramer MS & Krassioukov A V (2010). Care of rats with complete high-thoracic spinal cord injury. J Neurotrauma 27, 1709–1722. Resnick, R. B., R. S. Kestenbaum, and L. K. Schwartz. 1977. “Acute Systemic Effects of Cocaine in Man: A Controlled Study by Intranasal and Intravenous Routes.” Science (New York, N.Y.) 195(4279):696–98.  Roman GC. (2004). Brain hypoperfusion: a critical factor in vascular dementia. Neurological research 26, 454-458.  Rose, Kathryn M., Marsha L. Eigenbrodt, Rebecca L. Biga, David J. Couper, Kathleen C. Light, A. Richey Sharrett, and Gerardo Heiss. 2006. “Orthostatic Hypotension Predicts Mortality in Middle-Aged Adults: The Atherosclerosis Risk In Communities (ARIC) Study.” Circulation 114(7):630–36.  Rothwell, Peter M., Sally C. Howard, Eamon Dolan, Eoin O’Brien, Joanna E. Dobson, Bjorn Dahlöf, Peter S. Sever, and Neil R. Poulter. 2010. “Prognostic Significance of Visit-to-Visit Variability, Maximum Systolic Blood Pressure, and Episodic Hypertension.” Lancet (London, England) 375(9718):895–905.  Rosendorff C (1996). The renin-angiotensin system and vascular hypertrophy. J Am Coll Cardiol 28, 803–812. Roshan Moniri N (2012). Vascular changes in spinal cord injured animals with repetitive episodes of autonomic dysreflexia. Ryan, K. L., C. A. Rickards, C. Hinojosa-Laborde, W. H. Cooke, and V. A. Convertino. 2011.  76 “Arterial Pressure Oscillations Are Not Associated with Muscle Sympathetic Nerve Activity in Individuals Exposed to Central Hypovolaemia.” J Physiol 589(Pt 21):5311–22.  Sachdeva R, Gao F, Chan CCH & Krassioukov AV. (2018). Cognitive function after spinal cord injury: A systematic review. Neurology 91, 611-621.  Sachdeva R, Nightingale TE & Krassioukov AV. (2019). The Blood Pressure Pendulum following Spinal Cord Injury: Implications for Vascular Cognitive Impairment. Int J Mol Sci 20.  Sato K, Fisher JP, Seifert T, Overgaard M, Secher NH & Ogoh S (2012). Blood flow in internal carotid and vertebral arteries during orthostatic stress. Exp Physiol 97, 1272–1280. Satoh C, Fukuda N, Hu WY, Nakayama M, Kishioka H & Kanmatsuse K (2001). Role of endogenous angiotensin II in the increased expression of growth factors in vascular smooth muscle cells from spontaneously hypertensive rats. J Cardiovasc Pharmacol 37, 108–118. Scott JM, Warburton DE, Williams D, Whelan S & Krassioukov A (2011). Challenges, concerns and common problems: physiological consequences of spinal cord injury and microgravity. Spinal Cord 49, 4–16. Sofronova SI, Tarasova OS, Gaynullina D, Borzykh AA, Behnke BJ, Stabley JN, McCullough DJ, Maraj JJ, Hanna ME, Muller-Delp JM, Vinogradova OL & Delp MD (2015). Spaceflight on the Bion-M1 Biosatellite Alters Cerebral Artery Vasomotor and Mechanical Properties in Mice. J Appl Physioljap.00976.2014. Sukumaran SV, Singh TU, Parida S, Narasimha Reddy Ch E, Thangamalai R, Kandasamy K, Singh V & Mishra SK. (2013). TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery. Pharmacol Res 78, 18-27.  Taglialatela G, Hogan D, Zhang WR & Dineley KT. (2009). Intermediate- and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition. Behavioural brain research 200, 95-99.  Taylor CR & Levenson RM (2006). Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II. Histopathology 49, 411–424. Tuday EC, Nyhan D, Shoukas AA & Berkowitz DE (2009). Simulated microgravity-induced aortic remodeling. J Appl Physiol 106, 2002–2008. Tzeng, Yu-Chieh, Chris K. Willie, Greg Atkinson, Samuel J. E. E. Lucas, Aaron Wong, and Philip N. Ainslie. 2010. “Cerebrovascular Regulation during Transient Hypotension and Hypertension in Humans.” Hypertension 56(2):268–73.  Vlachopoulos, C., F. Kosmopoulou, D. Panagiotakos, N. Ioakeimidis, N. Alexopoulos, C. Pitsavos, and C. Stefanadis. 2004. “Smoking and Caffeine Have a Synergistic Detrimental Effect on Aortic Stiffness and Wave Reflections.” J Am Coll Cardiol 44(9):1911–17.  77  Wan D & Krassioukov A V (2014). Life-threatening outcomes associated with autonomic dysreflexia: a clinical review. J Spinal Cord Med 37, 2–10. Wang H, Luo W, Wang J, Guo C, Wang X, Wolffe SL, Bodary PF & Eitzman DT. (2012). Obesity-induced endothelial dysfunction is prevented by deficiency of P-selectin glycoprotein ligand-1. Diabetes 61, 3219-3227.  Wang Y, Yang Y, Wang A, An S, Li Z, Zhang W, Liu X, Ruan C, Liu X, Guo X, Zhao X & Wu S. (2016). Association of long-term blood pressure variability and brachial-ankle pulse wave velocity: a retrospective study from the APAC cohort. Sci Rep 6, 21303.  Wecht JM & Bauman WA (2013). Decentralized cardiovascular autonomic control and cognitive deficits in persons with spinal cord injury. J Spinal Cord Med 36, 74–81. Wecht JM, Radulovic M, Lafountaine MF, Rosado-Rivera D, Zhang RL & Bauman WA (2009). Orthostatic responses to nitric oxide synthase inhibition in persons with tetraplegia. Arch Phys Med Rehabil 90, 1428–1434. Wecht JM, Radulovic M, Weir JP, Lessey J, Spungen AM & Bauman WA (2005). Partial angiotensin-converting enzyme inhibition during acute orthostatic stress in persons with tetraplegia. J Spinal Cord Med 28, 103–108.  Wecht JM, Weir JP, Radulovic M & Bauman WA. (2016). Effects of midodrine and L-NAME on systemic and cerebral hemodynamics during cognitive activation in spinal cord injury and intact controls. Physiological Reports 4.  Wegener S, Wu WC, Perthen JE & Wong EC. (2007). Quantification of rodent cerebral blood flow (CBF) in normal and high flow states using pulsed arterial spin labeling magnetic resonance imaging. Journal of magnetic resonance imaging : JMRI 26, 855-862.  West CR, Popok D, Crawford M & Krassioukov A V (2015). Characterizing the temporal development of cardiovascular dysfunction in response to spinal cord injury. J Neurotrauma; DOI: 10.1089/neu.2014.3722. West, Christopher R., Mark A. Crawford, Malihe-Sadat Poormasjedi-Meibod, Katharine D. Currie, Andre Fallavollita, Violet Yuen, John H. McNeill, and Andrei V Krassioukov. 2014. “Passive Hind-Limb Cycling Improves Cardiac Function and Reduces Cardiovascular Disease Risk in Experimental Spinal Cord Injury.” The Journal of Physiology 592:1771–83. Wiesmann M, Kiliaan AJ & Claassen JAHR (2013). Vascular aspects of cognitive impairment and dementia. J Cereb Blood Flow Metab 33, 1696–1706. Willie, Christopher K., Yu-Chieh Tzeng, Joseph A. Fisher, and Philip N. Ainslie. 2014. “Integrative Regulation of Human Brain Blood Flow.” The Journal of Physiology 592(5):841–59.  78 Wilson LC, Cotter JD, Fan JL, Lucas RAI, Thomas KN & Ainslie PN (2010). Cerebrovascular reactivity and dynamic autoregulation in tetraplegia. Am J Physiol Integr Comp Physiol 298, R1035–R1042. Wu J, Stoica BA, Luo T, Sabirzhanov B, Zhao Z, Guanciale K, Nayar SK, Foss CA, Pomper MG & Faden AI. (2014). Isolated spinal cord contusion in rats induces chronic brain neuroinflammation, neurodegeneration, and cognitive impairment. Involvement of cell cycle activation. Cell cycle 13, 2446-2458.  Wu JC, Chen YC, Liu L, Chen TJ, Huang WC, Cheng H & Tung-Ping S (2012). Increased risk of stroke after spinal cord injury: a nationwide 4-year follow-up cohort study. Neurology 78, 1051–1057. Yang, S. T., W. G. Mayhan, F. M. Faraci, and D. D. Heistad. 1991. “Endothelium-Dependent Responses of Cerebral Blood Vessels during Chronic Hypertension.” Hypertension 17(5):612–18. Yu QJ, Tao H, Wang X & Li MC. (2015). Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke. Neural Regen Res 10, 1882-1891.   Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, McQueen M, Budaj A, Pais P, Varigos J & Lisheng L. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet 364, 937–952. Zhang DX & Gutterman DD. (2011). Transient receptor potential channel activation and endothelium-dependent dilation in the systemic circulation. J Cardiovasc Pharmacol 57, 133-139.  Zhang LN, Zhang LF & Ma J (2001). Simulated microgravity enhances vasoconstrictor responsiveness of rat basilar artery. J Appl Physiol 90, 2296–2305. Zieman SJ, Melenovsky V & Kass DA (2005). Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler Thromb Vasc Biol 25, 932–943.   

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